Dkk1 antibodies and methods of use

ABSTRACT

The present invention provides antibodies and immunologically functional fragments thereof that specifically bind DKK1 polypeptides. Methods for preparing such antibodies or fragments thereof as well as physiologically acceptable compositions containing the antibodies or fragments are also provided. Use of the antibodies and fragments to treat various diseases are also disclosed.

This application is a continuation of U.S. application Ser. No.13/878,619, filed Apr. 10, 2013, which is a U.S. National StageApplication of PCT Application No. PCT/US11/58025, filed Oct. 27, 2011,which claims the benefit of U.S. Provisional Application No. 61/407,128,filed Oct. 27, 2010, each of which is hereby incorporated by reference.

The present application is being filed along with a sequence listing inelectronic format. The sequence listing is provided as a file entitledA-1574-US-CNT-SeqListRev121117_ST25.txt, created Dec. 13, 2017, which is123.71 kb in size. The information in the electronic format of thesequence listing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to selective binding agents for dickkopf-1 (DKK1)protein, and more particularly, to antibodies and antigen bindingdomains and CDR regions that mediate selective binding to DKK1 proteins.

BACKGROUND OF THE INVENTION

Two or three distinct phases of changes to bone mass occur over the lifeof an individual (see Riggs, West J. Med. 154:63 77 (1991)). The firstphase occurs in both men and women and proceeds to attainment of a peakbone mass. This is achieved through linear growth of the endochondralgrowth plates and radial growth due to a rate of periosteal apposition.The second phase begins around age 30 for trabecular bone (flat bonesmore commonly found in the vertebrae and pelvis) and about age 40 forcortical bone (e.g., predominantly found in long bones such as in thelimbs) and continues to old age. This phase is characterized by slowbone loss and occurs in both men and women. In women, a third phase ofbone loss also occurs, most likely due to postmenopausal estrogendeficiencies. During this phase alone, women may lose an additional bonemass from the cortical bone and from the trabecular compartment (seeRiggs, supra).

Loss of bone mineral content can be caused by a wide variety ofconditions and may result in significant medical problems. For example,osteoporosis is a debilitating disease in humans and is characterized bymarked decreases in skeletal bone mass and mineral density, structuraldeterioration of bone, including degradation of bone microarchitectureand corresponding increases in bone fragility (i.e., decreases in bonestrength), and susceptibility to fracture in afflicted individuals.Osteoporosis in humans is generally preceded by clinical osteopenia(bone mineral density that is greater than one standard deviation butless than 2.5 standard deviations below the mean value for young adultbone), a condition found in approximately 25 million people in theUnited States. Another 7 8 million patients in the United States havebeen diagnosed with clinical osteoporosis (defined as bone mineralcontent greater than 2.5 standard deviations below that of mature youngadult bone). The frequency of osteoporosis in the human populationincreases with age. Among Caucasians, osteoporosis is predominant inwomen who, in the United States, comprise 80% of the osteoporosispatient pool. The increased fragility and susceptibility to fracture ofskeletal bone in the aged is aggravated by the greater risk ofaccidental falls in this population. Fractured hips, wrists, andvertebrae are among the most common injuries associated withosteoporosis. Hip fractures in particular are extremely uncomfortableand expensive for the patient, and for women, correlate with high ratesof mortality and morbidity.

Although osteoporosis has been regarded as an increase in the risk offracture due to decreased bone mass, few of the presently availabletreatments for skeletal disorders can increase the bone density ofadults, and most of the presently available treatments work primarily byinhibiting further bone resorption rather than stimulating new boneformation. Estrogen is now being prescribed to retard bone loss.However, some controversy exists over whether patients gain any longterm benefit and whether estrogen has any effect on patients over 75years old. Calcitonin, osteocalcin with vitamin K, or high doses ofdietary calcium, with or without vitamin D, have also been suggested forpostmenopausal women. High doses of calcium, however, often haveundesired gastrointestinal side effects, and serum and urinary calciumlevels must be continuously monitored (e.g., Khosla and Riggs, MayoClin. Proc. 70:978982, 1995).

Other current therapeutic approaches to osteoporosis includebisphosphonates (e.g., Fosamax™, Actonel™, Bonviva™, Zometa™,olpadronate, neridronate, skelid, bonefos), parathyroid hormone,calcilytics, anabolic steroids, lanthanum and strontium salts, andsodium fluoride. Such therapeutics, however, are often associated withundesirable side effects (see Khosla and Riggs, supra).

Dickkopf-1 (DKK1) is a member of the dickkopf family of proteins thathave been shown to be negative regulators of Wnt-signaling, which has acentral role in bone development and formation (see, e.g., Glinka etal., Nature 391:357-62 (1998); Fedi et al., J Biol Chem 274(27):19465-72(1999); Zorn, Curr Biol 11:R592-95 (2001); and Krupnik et al., Gene 238:301-13 (1999)). DKK1 inhibits Wnt signaling through its interaction withthe Wnt co-receptors LRP5 or LRP6 and the kremen proteins (see, forexample, Bafico et al., Nature Cell Biol 3:683 (2001); Mao et al.,Nature 411(17):321 (2001); Mao et al., Nature 417:664 (2002); andSemenov et al., Curr Biol 11:951-61 (2001). By binding LRP5 (LRP6) andkremen proteins, DKK1 prevents LRP5 or LRP6 from associating withmembers of the Wnt pathway and thus prevents Wnt-mediated signaltransduction, which in turn results in the inhibition of bone formation.

The DKK1 receptor LRP5/6 is a key protein in regulating bone mass (see,for example, Gong et al., Cell 107:513-23 (2001); Patel, N Eng J Med346(20):1572 (2002)). An autosomal recessive disorder characterized bylow bone mass (osteoporosis-pseudoglioma syndrome, or “OPPG”) has beenidentified as being caused by loss-of-function mutations in LRP5 (Gonget al., 2001). In addition, gain-of-function mutations in LRP5 have beenshown to result in autosomal dominant high bone mass in humans (Littleet al., Am J Human Genetics. 70(1):11-19, 2002). The same mutations inLRP5 that result in high bone mass can interfere with the ability ofDKK1 to inhibit LRP5 signaling (see, for example, Boyden et al., N Eng JMed. 346(20):1513-1521, 2002). Thus, DKK1 is appropriately characterizedas being a negative regulator of bone deposition.

Sclerostin, the product of the SOST gene, is absent in sclerosteosis, askeletal disease illustrated by bone overgrowth and strong dense bones(Brunkow et al., Am. J. Hum. Genet., 68:577 589, 2001; Balemans et al.,Hum. Mol. Genet., 10:537 543, 2001). Inhibitors of sclerostin have beenshown to increase the rate of bone mineralization, and thus bone mineraldensity (Padhi et al., J Bone Miner Res. 2010 June; epublished ahead ofprint). Likewise, DKK1 has been shown to be involved in the regulationof bone formation, particularly in bone fracture repair, and its role invarious other diseases that are associated with bone loss (e.g., cancerand diabetes).

Given the drawbacks of current therapies there is a need for improvedtherapeutics in the area of bone loss, such as osteoporosis, andimproved fracture repair among other bone disorders.

SUMMARY OF THE INVENTION

Provided herein are novel DKK1 inhibitors that are effective in treatingconditions requiring increased bone building, for example, fracturerepair or bone loss associated with pathological conditions, such asmultiple myeloma. In addition, provided herein are combinations ofagents that increase bone anabolism including combinations of DKK1 andsclerostin inhibitors. These combinations can be used for treatment of,for example, osteoporosis, accelerating healing of fractures, and anynumber of conditions requiring an increase in the rate of bone building.The combination can be two separate inhibitors, for example, ananti-sclerostin antibody and an anti-DKK1 antibody, or can be a singlemolecular entity, for example, a bispecific molecule including abispecific antibody.

Also provided herein are a variety of antibodies that bind DKK1. Theanti-DKK1 agents may also block or reduce binding between DKK1 and LRP5and/or LRP6, thereby stimulating at least one activity associated withWnt signaling. The agents can be an antibody or an immunologicallyfunctional fragment thereof and thus include antibodies with a naturallyoccurring structure, as well as polypeptides that have an antigenbinding domain (e.g., a domain antibody). The antibodies and fragmentscan be used to treat a variety of different diseases includingpreventing or treating conditions relating to loss of bone mass or tostimulate production of new bone, as well as various non-bone relateddisorders. Nucleic acids molecules, vectors, and host cells useful inthe production of the antibodies and selective binding agents are alsoprovided.

Some of the antibodies and immunologically functional fragments that areprovided include one or more of the following light chain (LC)complementary determining regions (CDRs): (i) a LC CDR1 with at least80% sequence identity to SEQ ID NO: 97, 103, 109, 115, 121, 127, 133,139, 145, 151, 157, 163, 169, 175, 181, 187, 193, 199, 205, 211, 217, or223; (ii) a LC CDR2 with at least 80% sequence identity to SEQ ID NO:98, 104, 110, 116, 122, 128, 134, 139, 146, 152, 158, 164, 170, 176,182, 188, 194, 200, 206, 212, 218, or 224; and (iii) a LC CDR3 with atleast 80% sequence identity to SEQ ID NO: 99, 105, 111, 117, 123, 129,135, 140, 147, 153, 159, 165, 171, 177, 183, 189, 195, 201, 207, 213,219, or 225. Some of the antibodies and immunologically functionalfragments that are provided include one or more of the preceding LC CDRsand/or one or more of the following heavy chain (HC) complementarydetermining regions (CDRs): (i) a HC CDR1 with at least 80% sequenceidentity to SEQ ID NO: 100, 106, 112, 118, 124, 130, 136, 142, 148, 154,160, 166, 172, 178, 184, 190, 196, 202, 208, 214, 220, or 226; (ii) a HCCDR2 with at least 80% sequence identity to SEQ ID NO: 101, 107, 113,119, 125, 131, 137, 143, 149, 155, 161, 167, 173, 179, 185, 191, 197,203, 209, 215, 221, or 227; and (iii) a HC CDR3 with at least 80%sequence identity to SEQ ID NO: 102, 108, 114, 120, 126, 132, 138, 144,150, 156, 162, 168, 174, 180, 186, 192, 198, 204, 210, 216, 222 or 228.Some of the antibodies and immunologically functional fragments thereofthat are provided also include one or more LC CDRs and one or more HCCDRs above.

Such antibodies or fragments can specifically bind a DKK1 polypeptide.Certain antibodies or fragments include one, two, three, four, five orall six of the forgoing CDRs.

The light chain and heavy chains of other antibodies or fragments are asdescribed above but have at least 90% sequence identity to the foregoingsequences. Still other antibodies or fragments thereof are ones having alight chain in which CDR1 has the amino acid sequence as set forth inSEQ ID NOs: 97, 103, 109, 115, 121, 127, 133, 139, 145, 151, 157, 163,169, 175, 181, 187, 193, 199, 205, 211, 217, or 223, CDR2 has the aminoacid sequence as set forth in SEQ ID NOs: 98, 104, 110, 116, 122, 128,134, 139, 146, 152, 158, 164, 170, 176, 182, 188, 194, 200, 206, 212,218, or 224 and/or CDR3 has the amino acid sequence as set forth in SEQID NOs: 99, 105, 111, 117, 123, 129, 135, 140, 147, 153, 159, 165, 171,177, 183, 189, 195, 201, 207, 213, 219, or 225. Some antibodies andfragments may also have a heavy chain in which CDR1 has the amino acidsequence as set forth in SEQ ID NOs: 100, 106, 112, 118, 124, 130, 136,142, 148, 154, 160, 166, 172, 178, 184, 190, 196, 202, 208, 214, 220, or226, CDR2 has the amino acid sequence as set forth in SEQ ID NOs: 101,107, 113, 119, 125, 131, 137, 143, 149, 155, 161, 167, 173, 179, 185,191, 197, 203, 209, 215, 221, or 227 and/or HC CDR3 has the amino acidsequence as set forth in SEQ ID NOs: 102, 108, 114, 120, 126, 132, 138,144, 150, 156, 162, 168, 174, 180, 186, 192, 198, 204, 210, 216, 222 or228. Certain antibodies or fragments include a light chain CDR3 with theamino acid sequence of SEQ ID NOs: 99, 105, 111, 117, 123, 129, 135,140, 147, 153, 159, 165, 171, 177, 183, 189, 195, 201, 207, 213, 219, or225 and/or a heavy chain CDR3 with the amino acid sequence of SEQ IDNOs: 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174,180, 186, 192, 198, 204, 210, 216, 222 or 228.

Certain other antibodies and immunologically functional fragments thatare provided include (a) a light chain variable region (VL) having 80%,85%, 90%, 92%, 95% or greater sequence identity with SEQ ID NO: 10, 14,18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86,90, or 94; (b) a heavy chain variable region (VH) having at least 80%sequence identity with SEQ ID NO: 12, 16, 20, 24, 28, 32, 36, 40, 44,48, 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, or 96; or (c) a VL of(a) and a VH of (b).

Other antibodies or fragments are similar in structure but the VL has atleast 90%, 92%, or more preferably 95% sequence identity with SEQ IDNOs: 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74,78, 82, 86, 90, or 94; and the VH has at least 90% sequence identitywith SEQ ID NOs: 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64,68, 72, 76, 80, 84, 88, 92, or 96. In certain antibodies or fragments,the VL has at least 98% sequence identity with SEQ ID NO:84, 28 or 32;and the VH has at least 98% sequence identity with SEQ ID NO: 10, 14,18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86,90, or 94. Still other antibodies or fragments are ones that include aVL that has the amino acid sequence of SEQ ID NO: 10, 14, 18, 22, 26,30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, or 94,and/or a VH that has the amino acid sequence of SEQ ID NOs: 12, 16, 20,24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92,or 96.

Some antibodies or fragments include a light chain that comprises orconsists of the amino acid sequence of SEQ ID NOs: 10, 14, 18, 22, 26,30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, or 94and/or a heavy chain that comprises or consists of the amino acidsequence of SEQ ID NOs: 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56,60, 64, 68, 72, 76, 80, 84, 88, 92, or 96.

Also included are isolated antibodies or an immunologically functionalfragments thereof that specifically bind a mature human DKK1 proteinexpressed from the sequence depicted in SEQ ID NO: 1, wherein saidantibody binds to an epitope comprising two loops, said loops beingformed by disulfide bonds between amino acids 220 and 237 of SEQ ID NO:2 and between cysteine residues 245 and 263 of SEQ ID NO:2.

Other antibodies or fragments that are disclosed compete with anantibody such as those described above for specific binding to a DKK1polypeptide. For example, some antibodies and fragments compete with anantibody that consists of two identical heavy chains and two identicallight chains, wherein the heavy chains comprise SEQ ID NO: 42 and saidlight chains comprise SEQ ID NO: 44.

The various antibodies and fragments that are provided may include asingle light and/or heavy chain or a single variable light domain and/ora single variable heavy domain. Other antibodies and fragments includetwo light and/or two heavy chains. In those instances in which theantibody or fragment includes two light and/or heavy chains, the twolight chains in some instances are identical to one another; likewise,the two heavy chains in some instances are identical. The antibodiesthat are provided may include, for example, monoclonal antibodies, ahuman antibody, a chimeric antibody, or a humanized antibody. Theimmunologically functional fragments may include, but are not limitedto, a scFv, a Fab, a Fab′, a F(ab′)², or a domain antibody. In certaininstances, the antibody or fragment dissociates from a DKK1 polypeptidewith a k_(d) (k_(off)) of 5×10⁻⁴ or less.

Pharmaceutical compositions that include any of the foregoing antibodiesand immunologically active fragments are also provided. Suchcompositions typically also include a buffer, a pharmaceuticallyacceptable diluent, a carrier, a solubilizer, an emulsifier or apreservative. The use of the foregoing antibodies and immunologicallyactive fragments in the preparation of a pharmaceutical composition ormedicament is also provided.

A variety of nucleic acids encoding the foregoing antibodies are alsoprovided. Some nucleic acids, for instance, encode (a) a light chain CDRwith the amino acid sequence as set forth in SEQ ID NOs: 9, 13, 17, 21,25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89,and/or 93; and/or (b) a heavy chain CDR with the amino acid sequence asset forth in SEQ ID NOs: 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55,59, 63, 67, 71, 75, 79, 83, 87, 91 and/or 95, such that the encodedCDR(s) encode an antibody or an immunologically functional fragmentthereof that can specifically bind a DKK1 polypeptide. Certain othernucleic acids comprise or consist of a sequence that encodes a variablelight region (VL) and/or a variable heavy region (VH) of an antibody orimmunologically active fragment, wherein the VL has at least 80%, 90% or95% sequence identity with SEQ ID NOs: 9, 13, 17, 21, 25, 29, 33, 37,41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, or 93 and the VH hasat least 80% 90%, or 95% sequence identity with SEQ ID NOs: 11, 15, 19,23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, 87, 91or 95. Some of the nucleic acids include a sequence that encodes a VLthat comprises or consists of SEQ ID NOs: 9, 13, 17, 21, 25, 29, 33, 37,41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, or 93 and/or asequence that encodes a VH that comprises or consists of SEQ ID NOs: 11,15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83,87, 91 or 95. Expression vectors comprising the foregoing nucleic acidsare also disclosed herein, as are cells (e.g., CHO cells) that comprisesuch expression vectors. Methods of producing an antibody or animmunologically active fragment thereof by culturing cells that containsuch expression vectors are also described.

In another aspect, the use of the foregoing binding agents, e.g.,antibodies, or immunologically functional fragments or combinationthereof in the treatment of a variety of diseases is disclosed. Certainmethods, for instance, involve administering to a patient in needthereof an effective amount of an antibody or immunologically activefragment or combinations as described herein to bone trauma includingbut not limited to orthopedic procedures, dental procedures, implantsurgery, joint replacement, bone grafting, bone cosmetic surgery andbone repair such as fracture healing, nonunion healing, delayed unionhealing and facial reconstruction. One or more compositions may beadministered before, during and/or after the procedure, replacement,graft, surgery or repair or other disorders associated with bone damage.

Further provided herein are methods of treating or preventing loss ofbone mass comprising administering to a patient in need thereof atherapeutically effective amount of an antibody or immunologicallyfunctional fragment thereof as described herein (e.g., an antibody orimmunologically functional fragment that comprises at least one lightchain CDR selected from the group consisting of amino acids depicted inSEQ ID NOs: 97, 103, 109, 115, 121, 127, 133, 139, 145, 151, 157, 163,169, 175, 181, 187, 193, 199, 205, 211, 217, or 223, or amino acidsdepicted in SEQ ID NOs: 98, 104, 110, 116, 122, 128, 134, 139, 146, 152,158, 164, 170, 176, 182, 188, 194, 200, 206, 212, 218, or 224 and theamino acids depicted in SEQ ID NOs: 99, 105, 111, 117, 123, 129, 135,140, 147, 153, 159, 165, 171, 177, 183, 189, 195, 201, 207, 213, 219, or225, and/or at least one heavy chain CDR selected from the groupconsisting of the amino acids depicted in SEQ ID NOs: 100, 106, 112,118, 124, 130, 136, 142, 148, 154, 160, 166, 172, 178, 184, 190, 196,202, 208, 214, 220, or 226, amino acids depicted in SEQ ID NOs: 101,107, 113, 119, 125, 131, 137, 143, 149, 155, 161, 167, 173, 179, 185,191, 197, 203, 209, 215, 221, or 227 and the amino acids depicted in SEQID NOs: 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174,180, 186, 192, 198, 204, 210, 216, 222 or 228). In one aspect of thisembodiment, the patient is one who suffers from cancer that metastasizesto bone, and in another aspect, the patient is one who suffers frommultiple myeloma. In yet another aspect, the patient is selected frompatients who have osteoporosis, osteopenia, Paget's disease,periodontitis, rheumatoid arthritis, and bone loss due toimmobilization. In yet other embodiments, the patient is selected fromthose who have bone damage that may or may not result from an underlyingloss of bone mass such as that caused by osteoporosis or osteolyticlesions associated with cancer (e.g., multiple myeloma). Examples ofsuch bone damage include but are not limited to orthopedic procedures,dental procedures, implant surgery, joint replacement (e.g., hipreplacement, knee replacement, etc.), bone grafting, bone cosmeticsurgery and bone repair such as fracture healing, nonunion healing,delayed union healing and facial reconstruction. One or morecompositions may be administered before, during and/or after theprocedure, replacement, graft, surgery or repair.

In yet other embodiments, the patient is selected from those who havebone loss that may or may not result from a condition such as thatcaused by osteoporosis, osteolytic lesions associated with cancer (e.g.,multiple myeloma).

These and other aspects of the present invention will become apparentupon reference to the following detailed description and attacheddrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Epitope sites of human DKK1 antibodies. Trypsin sites areindicated with solid arrows and AspN sites with dotted line arrows.Trypsin sites are in solid arrows and AspN sites in dotted arrows. Thebinding region for Ab 5.25.1 includes two discontinuous portions, thefirst from amino acids 98 to 104 and a region from amino acids 107-121and 127-140. The last three disulfide bonds form a major epitope regionwhere all tryptic sites can be protected by Ab 5.25.1. ARG102 is alsoprotected from trypsin digestion. Removal of amino acids position121-125 by CNBr treatment does not cause loss of binding. The regionnoted as resistant to AspN digestion may not be accessed for antibodybinding.

FIG. 2: Panel A lane 1 only LRP6-His is included; lane 2 rhDKK1-Flag;lane 3 hLRP6-His+hDKK1-Flag; lane 4 hLRP6-His+hDKK1-Flag+5.80.1; lane 5hLRP6-His+hDKK1-Flag+6.37.5; lane 6 hLRP6-His+hDKK1-Flag+r11H10; lane 7hLRP6-His+hDKK1-Flag+5.25.1; lane 8 hLRP6-His+hDKK1-Flag+5.77.1. Panel Blane 1 only LRP6-His is included; lane 2 rhDKK1-Flag; lane 3hLRP6-His+hDKK1-Flag; lane 4 hLRP6-His+hDKK1-Flag+0.5 μg 5.80.1; lane 5hLRP6-His+hDKK1-Flag+5 μg 5.80.1; lane 6 hLRP6-His+hDKK1-Flag+0.5 μg6.37.5; lane 7 hLRP6-His+hDKK1-Flag+5 μg 6.37.5; lane 8hLRP6-His+hDKK1-Flag+0.5 μg r11H10; lane 9 hLRP6-His+hDKK1-Flag+5 μgr11H10; lane 10 hLRP6-His+hDKK1-Flag+5 μg 5.25.1; lane 11hLRP6-His+hDKK1-Flag+5 μg 5.77.1.

FIG. 3: Shows the percent change in tibia bone mineral density at threeweeks for vehicle, PTH and different amounts of antibody 2.40.2. The 20mg/kg dose was significantly different from the vehicle.

FIG. 4: Antibody 5.32.1 from the 5.25.1 bin and 5.80.1 from the 11H10bin were tested in vivo for their ability to increase osteocalcin.Eight-week-old male BDF-1 mice were injected subcutaneously over atwo-week period with one of three doses of the purified monoclonalantibody (3, 10, or 30 mg/kg). Six mice were used per group. Negativecontrol mice were injected with vehicle (PBS).

FIG. 5: Mice were injected subcutaneously twice per week for three weekswith 25 mg/kg of the respective antibodies (6.37.5 and 6.116.6). Tenmice were used per group. Control groups were injected with vehicle(twice per week) or PTH (100 μg/kg five times per week). The data arepresented as percent change from baseline in bone mineral density of thelumbar vertebrae.

FIG. 6: An additional study was conducted with the rat 11H10 binantibody in a rat closed fracture healing model. The fully rat 11H10 binantibody r11H10 was utilized in this study as a surrogate molecule tothe fully human antibodies described herein. Improvement in maximum loadand BMD achieved with Anti-DKK1 treatment at the fracture callusindicating the acceleration of fracture healing.

FIG. 7: DKK1 in serum isolated from animal models of disease wasdetected and DKK1 protein levels are approximately five-fold elevated at3-weeks after induction of kidney damage with the pharmacological agent.

DETAILED DESCRIPTION

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclatures used in connection with, and techniques of, cell andtissue culture, molecular biology, immunology, microbiology, geneticsand protein and nucleic acid chemistry and hybridization describedherein are those well known and commonly used in the art. The methodsand techniques of the present invention are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification unless otherwiseindicated. See, e.g., Sambrook et al. Molecular Cloning: A LaboratoryManual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y. (1989) and Ausubel et al., Current Protocols in Molecular Biology,Greene Publishing Associates (1992), and Harlow and Lane Antibodies: ALaboratory Manual Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1990). Enzymatic reactions and purification techniques areperformed according to manufacturer's specifications, as commonlyaccomplished in the art or as described herein. The terminology used inconnection with, and the laboratory procedures and techniques of,analytical chemistry, synthetic organic chemistry, and medicinal andpharmaceutical chemistry described herein are those well known andcommonly used in the art. Standard techniques can be used for chemicalsyntheses, chemical analyses, pharmaceutical preparation, formulation,and delivery, and treatment of patients.

The following terms utilized in this disclosure, unless otherwiseindicated, will be understood to have the following meanings:

“DKK1” as used herein includes, for example, rat, murine, cynomolgousand human native forms of DKK1. Exemplary nucleotide sequences encodinghuman, murine, rat and cynomolgous DKK1 proteins are shown,respectively, in SEQ ID NOs: 1, 3, 5 and 7; the corresponding amino acidsequences are shown, respectively, in SEQ ID NOs: 2, 4, 6, and 8. Thehuman DKK1 protein (SEQ ID NO: 2) has a leader sequence consisting ofamino acids 1-31 of SEQ ID NO: 2. An exemplary rat DKK1 protein sequenceis listed in GenBank Accession XP219804. The term also includes variantsof such native sequences that are immunologically cross-reactive withthese native proteins. These proteins can inhibit the interactionbetween LRP5 or LRP6 with Wnt. The term can also refer to a fragment ofa native or variant form of DKK1 that contains an epitope to which anantibody can specifically bind.

The term “polynucleotide” or “nucleic acid” means single-stranded ordouble-stranded polymers. The nucleotides comprising the polynucleotidecan be ribonucleotides or deoxyribonucleotides or a modified form ofeither type of nucleotide. Said modifications include base modificationssuch as bromouridine and inosine derivatives, ribose modifications suchas 2′,3′-dideoxyribose, and internucleotide linkage modifications suchas phosphorothioate, phosphorodithioate, phosphoroselenoate,phosphorodiselenoate, phosphoroanilothioate, phoshoraniladate andphosphoroamidate. The term includes both single and double strandedforms.

The term “oligonucleotide” means a polynucleotide comprising 200 orfewer nucleotides. In some embodiments, oligonucleotides are 10 to 60bases in length. In other embodiments, oligonucleotides are 12, 13, 14,15, 16, 17, 18, 19, or 20 to 40 nucleotides in length. Oligonucleotidesmay be single stranded or double stranded, e.g., for use in theconstruction of a mutant gene.

An “isolated nucleic acid molecule” means a DNA or RNA of genomic, mRNA,cDNA, or synthetic origin or some combination thereof which is notassociated with all or a portion of a polynucleotide in which theisolated polynucleotide is found in nature, or is linked to apolynucleotide to which it is not linked in nature. For purposes of thisdisclosure, it should be understood that “a nucleic acid moleculecomprising” a particular nucleotide sequence does not encompass intactchromosomes. Isolated nucleic acid molecules “comprising” specifiednucleic acid sequences may include, in addition to the specifiedsequences, coding sequences for up to ten or even up to twenty otherproteins or portions thereof, or may include operably linked regulatorysequences that control expression of the coding region of the recitednucleic acid sequences, and/or may include vector sequences.

Unless specified otherwise, the left-hand end of any single-strandedpolynucleotide sequence discussed herein is the 5′ end; the left-handdirection of double-stranded polynucleotide sequences is referred to asthe 5′ direction. The direction of 5′ to 3′ addition of nascent RNAtranscripts is referred to as the transcription direction; sequenceregions on the DNA strand having the same sequence as the RNA transcriptthat are 5′ to the 5′ end of the RNA transcript are referred to as“upstream sequences”; sequence regions on the DNA strand having the samesequence as the RNA transcript that are 3′ to the 3′ end of the RNAtranscript are referred to as “downstream sequences”.

The term “control sequence” refers to a polynucleotide sequence that canaffect the expression and processing of coding sequences to which it isligated. The nature of such control sequences may depend upon the hostorganism. In particular embodiments, control sequences for prokaryotesmay include a promoter, a ribosomal binding site, and a transcriptiontermination sequence. For example, control sequences for eukaryotes mayinclude promoters comprising one or a plurality of recognition sites fortranscription factors, transcription enhancer sequences, andtranscription termination sequence. “Control sequences” according to theinvention can include leader sequences and/or fusion partner sequences.

The term “vector” means any molecule or entity (e.g., nucleic acid,plasmid, bacteriophage or virus) used to transfer protein codinginformation into a host cell.

The term “expression vector” or “expression construct” refers to avector that is suitable for transformation of a host cell and containsnucleic acid sequences that direct and/or control (in conjunction withthe host cell) expression of one or more heterologous coding regionsoperatively linked thereto. An expression construct may include, but isnot limited to, sequences that affect or control transcription,translation, and, if introns are present, affect RNA splicing of acoding region operably linked thereto.

As used herein, “operably linked” means that the components to which theterm is applied are in a relationship that allows them to carry outtheir inherent functions under suitable conditions. For example, acontrol sequence in a vector that is “operably linked” to a proteincoding sequence is ligated thereto so that expression of the proteincoding sequence is achieved under conditions compatible with thetranscriptional activity of the control sequences.

The term “host cell” means a cell that has been transformed, or iscapable of being transformed, with a nucleic acid sequence and therebyexpresses a gene of interest. The term includes the progeny of theparent cell, whether or not the progeny is identical in morphology or ingenetic make-up to the original parent cell, so long as the gene ofinterest is present.

The term “transduction” means the transfer of genes from one bacteriumto another, usually by bacteriophage. “Transduction” also refers to theacquisition and transfer of eukaryotic cellular sequences byretroviruses.

The term “transfection” means the uptake of foreign or exogenous DNA bya cell, and a cell has been “transfected” when the exogenous DNA hasbeen introduced inside the cell membrane. A number of transfectiontechniques are well known in the art and are disclosed herein. See,e.g., Graham et al., 1973, Virology 52:456; Sambrook et al., 2001,Molecular Cloning: A Laboratory Manual, Id.; Davis et al., 1986, BasicMethods in Molecular Biology, Elsevier; and Chu et al., 1981, Gene13:197. Such techniques can be used to introduce one or more exogenousDNA moieties into suitable host cells.

The term “transformation” refers to a change in a cell's geneticcharacteristics, and a cell has been transformed when it has beenmodified to contain new DNA or RNA. For example, a cell is transformedwhere it is genetically modified from its native state by introducingnew genetic material via transfection, transduction, or othertechniques. Following transfection or transduction, the transforming DNAmay recombine with that of the cell by physically integrating into achromosome of the cell, or may be maintained transiently as an episomalelement without being replicated, or may replicate independently as aplasmid. A cell is considered to have been “stably transformed” when thetransforming DNA is replicated with the division of the cell.

The terms “polypeptide” or “protein” means a macromolecule having theamino acid sequence of a native protein, that is, a protein produced bya naturally-occurring and non-recombinant cell, or produced by agenetically-engineered or recombinant cell, and comprise moleculeshaving the amino acid sequence of the native protein, or moleculeshaving deletions from, additions to, and/or substitutions of one or moreamino acids of the native sequence. The terms “polypeptide” and“protein” specifically encompass anti-DKK1 antibodies, or sequences thathave deletions from, additions to, and/or substitutions of one or moreamino acid of anti-DKK1 antibody. The term “polypeptide fragment” refersto a polypeptide that has an amino-terminal deletion, acarboxyl-terminal deletion, and/or an internal deletion as compared withthe full-length native protein. Such fragments may also contain modifiedamino acids as compared with the native protein. In certain embodiments,fragments are about 5 to 500 amino acids long. For example, fragmentsmay be at least 5, 6, 8, 10, 14, 20, 50, 70, 100, 110, 150, 200, 250,300, 350, 400, or 450 amino acids long. Useful polypeptide fragments forthis invention include immunologically functional fragments ofantibodies, including binding domains. In the case of anti-DKK1antibody, useful fragments include but are not limited to a CDR region,a variable domain of a heavy or light chain, a portion of an antibodychain or just its variable region including two CDRs, and the like.

The term “isolated protein” referred to herein means that a subjectprotein (1) is free of at least some other proteins with which it wouldnormally be found, (2) is essentially free of other proteins from thesame source, e.g., from the same species, (3) is expressed by a cellfrom a different species, (4) has been separated from at least about 50percent of polynucleotides, lipids, carbohydrates, or other materialswith which it is associated in nature, (5) is operably associated (bycovalent or noncovalent interaction) with a polypeptide with which it isnot associated in nature, or (6) does not occur in nature. Genomic DNA,cDNA, mRNA or other RNA, of synthetic origin, or any combination thereofmay encode such an isolated protein. Preferably, the isolated protein issubstantially free from proteins or polypeptides or other contaminantsthat are found in its natural environment that would interfere with itstherapeutic, diagnostic, prophylactic, research or other use.

A “variant” of a polypeptide (e.g., an antibody) comprises an amino acidsequence wherein one or more amino acid residues are inserted into,deleted from and/or substituted into the amino acid sequence relative toanother polypeptide sequence. Variants of the invention include fusionproteins.

A “derivative” of a polypeptide is a polypeptide (e.g., an antibody)that has been chemically modified in some manner distinct frominsertion, deletion, or substitution variants, e.g., via conjugation toanother chemical moiety.

The term “antibody” refers to an intact immunoglobulin of any isotype,or a fragment thereof that can compete with the intact antibody forspecific binding to the target antigen, and includes chimeric,humanized, fully human, and bispecific antibodies. An intact antibodygenerally will comprise at least two full-length heavy chains and twofull-length light chains, but in some instances may include fewer chainssuch as antibodies naturally occurring in camelids which may compriseonly heavy chains. Antibodies according to the invention may be derivedsolely from a single source, or may be “chimeric,” that is, differentportions of the antibody may be derived from two different antibodies.For example, the CDR regions may be derived from a rat or murine source,while the framework region of the V region are derived from a differentanimal source, such as a human. The antibodies or binding fragments ofthe invention may be produced in hybridomas, by recombinant DNAtechniques, or by enzymatic or chemical cleavage of intact antibodies.Unless otherwise indicated, the term “antibody” includes, in addition toantibodies comprising two full-length heavy chains and two full-lengthlight chains, derivatives, variants, fragments, and muteins thereof,examples of which are described below.

The term “light chain” includes a full-length light chain and fragmentsthereof having sufficient variable region sequence to confer bindingspecificity. A full-length light chain includes a variable regiondomain, VL, and a constant region domain, CL. The variable region domainof the light chain is at the amino-terminus of the polypeptide. Lightchains according to the invention include kappa chains and lambdachains.

The term “heavy chain” includes a full-length heavy chain and fragmentsthereof having sufficient variable region sequence to confer bindingspecificity. A full-length heavy chain includes a variable regiondomain, VH, and three constant region domains, CH1, CH2, and CH3. The VHdomain is at the amino-terminus of the polypeptide, and the CH domainsare at the carboxyl-terminus, with the CH3 being closest to the —COOHend. Heavy chains according to the invention may be of any isotype,including IgG (including IgG1, IgG2, IgG3 and IgG4 subtypes), IgA(including IgA1 and IgA2 subtypes), IgM and IgE.

The term “immunologically functional fragment” (or simply “fragment”) ofan immunoglobulin chain, as used herein, refers to a portion of anantibody light chain or heavy chain that lacks at least some of theamino acids present in a full-length chain but which is capable ofbinding specifically to an antigen. Such fragments are biologicallyactive in that they bind specifically to the target antigen and cancompete with intact antibodies for specific binding to a given epitope.In one aspect of the invention, such a fragment will retain at least oneCDR present in the full-length light or heavy chain, and in someembodiments will comprise a single heavy chain and/or light chain orportion thereof. These biologically active fragments may be produced byrecombinant DNA techniques, or may be produced by enzymatic or chemicalcleavage of intact antibodies. Immunologically functional immunoglobulinfragments of the invention include, but are not limited to, Fab, Fab′,F(ab′)2, Fv, domain antibodies and single-chain antibodies, and may bederived from any mammalian source, including but not limited to human,mouse, rat, camelid or rabbit. It is contemplated further that afunctional portion of the inventive antibodies, for example, one or moreCDRs, could be covalently bound to a second protein or to a smallmolecule to create a therapeutic agent directed to a particular targetin the body, possessing bifunctional therapeutic properties, or having aprolonged serum half-life.

A “Fab fragment” is comprised of one light chain and the CH1 andvariable regions of one heavy chain. The heavy chain of a Fab moleculecannot form a disulfide bond with another heavy chain molecule.

An “Fc” region contains two heavy chain fragments comprising the CH2 andCH3 domains of an antibody and in some cases the lower hinge region. Thetwo heavy chain fragments are held together by two or more disulfidebonds (typically in the hinge region) and by hydrophobic interactions ofthe CH3 domains.

A “Fab′ fragment” contains one light chain and a portion of one heavychain that contains the VH domain and the CH1 domain and also the regionbetween the CH1 and CH2 domains, such that an interchain disulfide bondcan be formed between the two heavy chains of two Fab′ fragments to forma F(ab′)₂ molecule.

A “F(ab′)2 fragment” contains two light chains and two heavy chainscontaining a portion of the constant region between the CH1 and CH2domains, such that an interchain disulfide bond is formed between thetwo heavy chains. A F(ab′)2 fragment thus is composed of two Fab′fragments that are held together by a disulfide bond between the twoheavy chains.

The “Fv region” comprises the variable regions from both the heavy andlight chains, but lacks the constant regions.

“Single-chain antibodies” are Fv molecules in which the heavy and lightchain variable regions have been connected by a flexible linker to forma single polypeptide chain, which forms an antigen-binding region.Single chain antibodies are discussed in detail in International PatentApplication Publication No. WO 88/01649 and U.S. Pat. Nos. 4,946,778 and5,260,203.

A “domain antibody” is an immunologically functional immunoglobulinfragment containing only the variable region of a heavy chain or thevariable region of a light chain. In some instances, two or more VHregions are covalently joined with a peptide linker to create a bivalentdomain antibody. The two VH regions of a bivalent domain antibody maytarget the same or different antigens.

A “bivalent antibody” comprises two antigen binding sites. In someinstances, the two binding sites have the same antigen specificities.However, bivalent antibodies may be bispecific (see below).

A “multispecific antibody” is one that targets more than one antigen orepitope.

A “bispecific,” “dual-specific” or “bifunctional” antibody is a hybridantibody having two different antigen binding sites. Bispecificantibodies are a species of multispecific antibody and may be producedby a variety of methods including, but not limited to, fusion ofhybridomas or linking of Fab′ fragments. See, e.g., Songsivilai &Lachmann (1990), Clin. Exp. Immunol. 79:315-321; Kostelny et al. (1992),J. Immunol. 148:1547-1553. The two binding sites of a bispecificantibody will bind to two different epitopes, which may reside on thesame or different protein targets.

The term “neutralizing antibody” refers to an antibody that binds to aligand, prevents binding of the ligand to its binding partner andinterrupts the biological response that otherwise would result from theligand binding to its binding partner. In assessing the binding andspecificity of an antibody or immunologically functional fragmentthereof, an antibody or fragment will substantially inhibit binding of aligand to its binding partner when an excess of antibody reduces thequantity of binding partner bound to the ligand by at least about 20%,30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99% or more (asmeasured in an in vitro competitive binding assay). In the case ofantibodies to DKK1, a neutralizing antibody will diminish the ability ofDKK1 to bind LRP5 or LRP6, thereby inducing a measurable increase in Wntactivity.

The term “compete” when used in the context of antibodies that competefor the same epitope means competition between antibodies is determinedby an assay in which the antibody or immunologically functional fragmentunder test prevents or inhibits specific binding of a reference antibodyto a common antigen (e.g., DKK1 or a fragment thereof). Numerous typesof competitive binding assays can be used, for example: solid phasedirect or indirect radioimmunoassay (RIA), solid phase direct orindirect enzyme immunoassay (EIA), sandwich competition assay (see,e.g., Stahli et al. (1983) Methods in Enzymology 9:242-253); solid phasedirect biotin-avidin EIA (see, e.g., Kirkland et al., (1986) J. Immunol.137:3614-3619) solid phase direct labeled assay, solid phase directlabeled sandwich assay (see, e.g., Harlow and Lane (1988) Antibodies, ALaboratory Manual, Cold Spring Harbor Press); solid phase direct labelRIA using 1-125 label (see, e.g., Morel et al. (1988) Molec. Immunol.25:7-15); solid phase direct biotin-avidin EIA (see, e.g., Cheung, etal. (1990) Virology 176:546-552); and direct labeled RIA (Moldenhauer etal. (1990) Scand. J. Immunol. 32:77-82). Typically, such an assayinvolves the use of purified antigen bound to a solid surface or cellsbearing either of these, an unlabelled test immunoglobulin and a labeledreference immunoglobulin. Competitive inhibition is measured bydetermining the amount of label bound to the solid surface or cells inthe presence of the test immunoglobulin. Usually the test immunoglobulinis present in excess. Antibodies identified by competition assay(competing antibodies) include antibodies binding to the same epitope asthe reference antibody and antibodies binding to an adjacent epitopesufficiently proximal to the epitope bound by the reference antibody forsteric hindrance to occur. Additional details regarding methods fordetermining competitive binding are provided in the examples herein.Usually, when a competing antibody is present in excess, it will inhibitspecific binding of a reference antibody to a common antigen by at least40%, 45%, 50%, 55%, 60%, 65%, 70% or 75%. In some instance, binding isinhibited by at least 80%, 85%, 90%, 95%, or 97% or more by a selectivebinding agent, such as an antibody, and additionally capable of beingused in an animal to produce antibodies capable of binding to thatantigen. An antigen may possess one or more epitopes that are capable ofinteracting with different antibodies.

The term “epitope” includes any determinant capable of specificallybinding to an immunoglobulin or to a T-cell receptor. An epitope is aregion of an antigen that is bound by an antibody that specificallytargets that antigen, and when the antigen is a protein, includesspecific amino acids that directly contact the antibody. Most often,epitopes reside on proteins, but in some instances may reside on otherkinds of molecules, such as nucleic acids. Epitope determinants mayinclude chemically active surface groupings of molecules such as aminoacids, sugar side chains, phosphoryl or sulfonyl groups, and may havespecific three dimensional structural characteristics, and/or specificcharge characteristics. Generally, antibodies specific for a particulartarget antigen will preferentially recognize an epitope on the targetantigen in a complex mixture of proteins and/or macromolecules.

An antibody of the invention is said to “specifically bind” its targetantigen when the dissociation constant (K_(d)) is 1×10⁻⁷ M. The antibodyspecifically binds antigen with “high affinity” when the Kd is 1×10⁻⁸,higher affinity is M 5×10⁻⁹ M, and with “very high affinity” when the Kdis 5×10⁻¹⁰ M. In one embodiment of the invention, the antibody has a Kdof 1×10⁻⁹ M and an off-rate of about 1×10⁻⁴/sec. In one embodiment ofthe invention, the off-rate is <1×10⁻⁵. In other embodiments of theinvention, the antibodies will bind to human DKK1 with a Kd of betweenabout 1×10⁻⁸ M and 1×10⁻¹⁰ M, and in yet another embodiment it will bindwith a Kd 2×10⁻¹⁰. One of skill in the art will recognize thatspecifically binding does not mean exclusive binding, rather it allowsfor some degree of non-specific binding as is typical in biologicalreactions between groups with affinity to one another.

The term “identity” refers to a relationship between the sequences oftwo or more polypeptide molecules or two or more nucleic acid molecules,as determined by aligning and comparing the sequences. “Percentidentity” means the percent of identical residues between the aminoacids or nucleotides in the compared molecules and is calculated basedon the size of the smallest of the molecules being compared. For thesecalculations, gaps in alignments (if any) must be addressed by aparticular mathematical model or computer program (i.e., an“algorithm”). Methods that can be used to calculate the identity of thealigned nucleic acids or polypeptides include those described inComputational Molecular Biology, (Lesk, A. M., ed.), 1988, New York:Oxford University Press; Biocomputing Informatics and Genome Projects,(Smith, D. W., ed.), 1993, New York: Academic Press; Computer Analysisof Sequence Data, Part I, (Griffin, A. M., and Griffin, H. G., eds.),1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysisin Molecular Biology, New York: Academic Press; Sequence AnalysisPrimer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M.Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math. 48:1073.

In calculating percent identity, the sequences being compared arealigned in a way that gives the largest match between the sequences. Thecomputer program used to determine percent identity is the GCG programpackage, which includes GAP (Devereux et al., 1984, Nucl Acid Res12:387; Genetics Computer Group, University of Wisconsin, Madison,Wis.). The computer algorithm GAP is used to align the two polypeptidesor polynucleotides for which the percent sequence identity is to bedetermined. The sequences are aligned for optimal matching of theirrespective amino acid or nucleotide (the “matched span”, as determinedby the algorithm). A gap opening penalty (which is calculated as3.times. the average diagonal, wherein the “average diagonal” is theaverage of the diagonal of the comparison matrix being used; the“diagonal” is the score or number assigned to each perfect amino acidmatch by the particular comparison matrix) and a gap extension penalty(which is usually 1/10 times the gap opening penalty), as well as acomparison matrix such as PAM 250 or BLOSUM 62 are used in conjunctionwith the algorithm. In certain embodiments, a standard comparison matrix(see Dayhoff et al., 1978, Atlas of Protein Sequence and Structure5:345-352 for the PAM 250 comparison matrix; Henikoff et al., 1992,Proc. Natl. Acad. Sci. USA 89: 10915-10919 for the BLOSUM 62 comparisonmatrix) is also used by the algorithm.

Recommended parameters for determining percent identity for polypeptidesor nucleotide sequences using the GAP program are the following:

Algorithm: Needleman et al., 1970, J. Mol. Biol. 48:443-453;Comparison matrix: BLOSUM 62 from Henikoff et al., 1992, supra;Gap Penalty: 12 (but with no penalty for end gaps)

Gap Length Penalty: 4 Threshold of Similarity: 0

Certain alignment schemes for aligning two amino acid sequences mayresult in matching of only a short region of the two sequences, and thissmall aligned region may have very high sequence identity even thoughthere is no significant relationship between the two full-lengthsequences. Accordingly, the selected alignment method (GAP program) canbe adjusted if so desired to result in an alignment that spans at least50 contiguous amino acids of the target polypeptide.

As used herein, “substantially pure” means that the described species ofmolecule is the predominant species present, that is, on a molar basisit is more abundant than any other individual species in the samemixture. In certain embodiments, a substantially pure molecule is acomposition wherein the object species comprises at least 50% (on amolar basis) of all macromolecular species present. In otherembodiments, a substantially pure composition will comprise at least80%, 85%, 90%, 95%, or 99% of all macromolecular species present in thecomposition. In other embodiments, the object species is purified toessential homogeneity wherein contaminating species cannot be detectedin the composition by conventional detection methods and thus thecomposition consists of a single detectable macromolecular species.

The term “osteopenia” refers to a patient with bone loss of at least onestandard deviation compared with a standard patient considered to havenormal bone mineral density (BMD). For present purposes, the measurementis determined by Dual Energy X-ray Absorptiometry (DEXA) and thepatient's BMD is compared with an age and gender-matched standard (Zscore). In determining osteopenia, BMD measurements may be taken of oneor more bones.

The term “therapeutically effective amount” refers to the amount of ananti-DKK1 antibody determined to produce a therapeutic response in amammal. Such therapeutically effective amounts are readily ascertainedby one of ordinary skill in the art.

“Amino acid” includes its normal meaning in the art. The twentynaturally-occurring amino acids and their abbreviations followconventional usage. See Immunology—A Synthesis, 2nd Edition, (E. S.Golub and D. R. Gren, eds.), Sinauer Associates: Sunderland, Mass.(1991). Stereoisomers (e.g., D-amino acids) of the twenty conventionalamino acids, unnatural amino acids such as .alpha.-,.alpha.-disubstituted amino acids, N-alkyl amino acids, and otherunconventional amino acids may also be suitable components forpolypeptides of the invention. Examples of unconventional amino acidsinclude: 4-hydroxyproline, gamma-carboxyglutamate,.epsilon.-N,N,N-trimethyllysine, .epsilon.-N-acetyllysine,O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine,5-hydroxylysine, .sigma.-N-methylarginine, and other similar amino acidsand imino acids (e.g., 4-hydroxyproline). In the polypeptide notationused herein, the left-hand direction is the amino terminal direction andthe right-hand direction is the carboxyl-terminal direction, inaccordance with standard usage and convention.

The present invention provides novel compositions comprising antibodiesand antigen-binding sites of immunoglobulins specific for DKK1 (e.g., apolypeptide consisting of amino acids 32 to 266 of SEQ ID NO: 2). Someof these antibodies and antibody fragments can cross-react with DKK1from several mammalian sources, including rat, mouse, cynomolgus monkeyand human DKK1. Some of the antibodies and fragments have higheraffinity for DKK1 from one species than another (e.g., some antibodiesand fragments have higher affinity for human DKK1 as compared to rat ormurine DKK1; other antibodies have higher affinity for rat or murineDKK1 as compared to human DKK1). The invention also provides novelneutralizing antibodies, including chimeric, humanized and humanantibodies, as well as antibodies and immunologically functionalfragments thereof that bind a conformational epitope in human DKK1.Nucleic acids encoding the antibodies and fragments are also disclosed,as well as methods for expressing the antibodies using these nucleicacids. In another aspect, the invention relates to molecules (e.g.,immunologically functional fragments and polypeptides) that are capableof exhibiting immunological binding properties of antibodyantigen-binding sites.

The antibodies and immunologically functional fragments that aredisclosed herein have a variety of utilities. Some of the antibodies andfragments, for instance, are useful in specific binding assays, affinitypurification of DKK1 or its ligands and in screening assays to identifyother antagonists of DKK1 activity. Certain of the antibodies can beused to treat various diseases that are associated with the activity ofDKK1. Some antibodies and fragments can thus be used in a variety oftreatments related to bone such as increasing bone mineral density,synthesis of new bone, treatment of systemic bone loss (e.g., boneerosions), bone repair, and treatments for various forms of arthritis.Some antibodies can also be used to increase osteoclast activity andinduce bone resorption. Certain of the antibodies and fragments that aredisclosed, however, can be used to treat a variety of diverse diseasesthat are unrelated to bone diseases. As described in greater detailbelow, examples of such diseases include those in which it is desirableto promote stem cell renewal (e.g., diabetes and diseases of themuscle), inflammatory diseases (e.g., Crohn's and inflammatory boweldisease), neurological diseases, ocular diseases, renal diseases, andvarious skin disorders.

A variety of selective binding agents useful for regulating the activityof DKK1 are provided. These agents include, for instance, antibodies andimmunologically functional fragments thereof that contain an antigenbinding domain (e.g., single chain antibodies, domain antibodies,immunoadhesions, and polypeptides with an antigen binding region) andspecifically bind to a DKK1 polypeptide (e.g., a human, rat and/ormurine DKK1 polypeptide). Some of the agents, for example, are useful ininhibiting the binding of DKK1 to LRP5 and/or LRP6, and can thus be usedto stimulate one or more activities associated with Wnt signaling.

Some of the binding agents that are provided have the structuretypically associated with naturally occurring antibodies. The structuralunits of these antibodies typically comprise one or more tetramers, eachcomposed of two identical couplets of polypeptide chains, though somespecies of mammals also produce antibodies having only a single heavychain. In a typical antibody, each pair or couplet includes onefull-length “light” chain (in certain embodiments, about 25 kDa) and onefull-length “heavy” chain (in certain embodiments, about 50-70 kDa).Each individual immunoglobulin chain is composed of several“immunoglobulin domains,” each consisting of roughly 90 to 110 aminoacids and expressing a characteristic folding pattern. These domains arethe basic units of which antibody polypeptides are composed. Theamino-terminal portion of each chain typically includes a variabledomain that is responsible for antigen recognition. The carboxy-terminalportion is more conserved evolutionarily than the other end of the chainand is referred to as the “constant region” or “C region.” Human lightchains generally are classified as kappa and lambda light chains, andeach of these contains one variable domain and one constant domain.Heavy chains are typically classified as mu, delta, gamma, alpha, orepsilon chains, and these define the antibody's isotype as IgM, IgD,IgG, IgA, and IgE, respectively. IgG has several subtypes, including,but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM subtypes includeIgM, and IgM2. IgA subtypes include IgA1 and IgA2. In humans, the IgAand IgD isotypes contain four heavy chains and four light chains; theIgG and IgE isotypes contain two heavy chains and two light chains; andthe IgM isotype contains five heavy chains and five light chains. Theheavy chain C region typically comprises one or more domains that may beresponsible for effector function. The number of heavy chain constantregion domains will depend on the isotype. IgG heavy chains, forexample, each contain three C region domains known as CH1, CH2 and CH3.The antibodies that are provided can have any of these isotypes andsubtypes. In certain embodiments of the invention, the anti-DKK1antibody is of the IgG1, IgG2 or IgG4 subtype.

In full-length light and heavy chains, the variable and constant regionsare joined by a “J” region of about 12 or more amino acids, with theheavy chain also including a “D” region of about 10 more amino acids.See, e.g., Fundamental Immunology, 2nd ed., Ch. 7 (Paul, W., ed.) 1989,New York: Raven Press. The variable regions of each light/heavy chainpair typically form the antigen binding site.

Variable regions of immunoglobulin chains generally exhibit the sameoverall structure, comprising relatively conserved framework regions(FR) joined by three hypervariable regions, more often called“complementarity determining regions” or CDRs. The CDRs from the twochains of each heavy chain/light chain pair mentioned above typicallyare aligned by the framework regions to form a structure that bindsspecifically with a specific epitope on the target protein (e.g., DKK1).From N-terminal to C-terminal, naturally-occurring light and heavy chainvariable regions both typically conform with the following order ofthese elements: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. A numberingsystem has been devised for assigning numbers to amino acids that occupypositions in each of these domains. This numbering system is defined inKabat Sequences of Proteins of Immunological Interest (1987 and 1991,National Institutes of Health, Bethesda, Md.), or Chothia & Lesk, 1987,J. Mol. Biol. 196: 901-917; Chothia et al., 1989, Nature 342: 878-883.

Provided in Table 1 are human (SEQ ID NOs: 1 and 2), mouse (SEQ ID NOs:3 and 4), rat (SEQ ID NOs: 5 and 6) and cynomolgous monkey (SEQ ID NOs:7 and 8) nucleic acid and protein DKK1 sequences, respectively. Alsoprovided are specific examples of the light and heavy chains ofantibodies that are provided herein and their corresponding nucleotideand amino acid sequences. The sequence identifiers are provided in theleft most column, sequences (nucleic acid or protein) in the middle andinternal designations for the sequences on the right most column. Inaddition, the respective CDR's are provided (SEQ ID NOs: 97-228).Vh=variable heavy chain; Vk=variable kappa light chain; Vl=variablelambda light chain.

TABLE 1 SEQ ID NO DNA or Protein   1ATGATGGCTCTGGGCGCAGCGGGAGCTACCCGGGTCTTTGTCGCGATGGTAGCGGCGGCTCTCGGC HUMANGGCCACCCTCTGCTGGGAGTGAGCGCCACCTTGAACTCGGTTCTCAATTCCAACGCTATCAAGAAC DKK1CTGCCCCCACCGCTGGGCGGCGCTGCGGGGCACCCAGGCTCTGCAGTCAGCGCCGCGCCGGGAATCCTGTACCCGGGCGGGAATAAGTACCAGACCATTGACAACTACCAGCCGTACCCGTGCGCAGAGGACGAGGAGTGCGGCACTGATGAGTACTGCGCTAGTCCCACCCGCGGAGGGGACGCAGGCGTGCAAATCTGTCTCGCCTGCAGGAAGCGCCGAAAACGCTGCATGCGTCACGCTATGTGCTGCCCCGGGAATTACTGCAAAAATGGAATATGTGTGTCTTCTGATCAAAATCATTTCCGAGGAGAAATTGAGGAAACCATCACTGAAAGCTTTGGTAATGATCATAGCACCTTGGATGGGTATTCCAGAAGAACCACCTTGTCTTCAAAAATGTATCACACCAAAGGACAAGAAGGTTCTGTTTGTCTCAGGTCATCAGACTGTGCCTCAGGATTGTGTTGTGATAGACACTTCTGGTCCAAGATCTGTAAACCTGTCCTGAAAGAAGGTCAAGTGTGTACCAAGCATAGGAGAAAAGGCTCTCATGGACTAGAAATATTCCAGCGTTGTTACTGTGGAGAAGGTCTGTCTTGCCGGATACAGAAAGATCACCATCAAGCCAGTAATTCTTCTAGGCTTCACACTTGTCAGAGACAC   2MMALGAAGAT RVFVAMVAAA LGGHPLLGVS ATLNSVLNSN AIKNLPPPLG GAAGHPGSAVSAAPGILYPG GNKYQTIDNY QPYPCAEDEE CGTDEYCASP TRGGDAGVQI CLACRKRRKRCMRHAMCCPG NYCKNGICVS SDQNHFRGEI EETITESFGN DHSTLDGYSR RTTLSSKMYHTKGQEGSVCL RSSDCASGLC CDRHFWSKIC KPVLKEGQV C  TKHRRKGSHG LEIFQR C YCGEGLS C RIQKD HHQASNSSRL HT C QRH   3ATGATGGTTGTGTGTGCAGCGGCAGCTGTCCGGTTCTTGGCCGTGTTTACAATGATGGCTCTCTGCMurineAGCCTCCCTCTGCTAGGAGCCAGTGCCACCTTGAACTCAGTTCTCATCAATTCCAACGCGATCAAG DKK1AACCTGCCCCCACCGCTGGGTGGTGCTGGGGGGCAGCCGGGCTCTGCTGTCAGTGTGGCGCCGGGAGTTCTCTATGAGGGCGGGAACAAGTACCAGACTCTTGACAACTACCAGCCCTACCCTTGCGCTGAAGATGAGGAGTGCGGCTCTGACGAGTACTGCTCCAGCCCCAGCCGCGGGGCAGCCGGCGTCGGAGGTGTACAGATCTGTCTGGCTTGCCGAAAGCGCAGGAAGCGCTGCATGAGGCACGCTATGTGCTGCCCCGGGAACTACTGCAAAAATGGAATATGCATGCCCTCTGACCACAGCCATTTTCCTCGAGGGGAGATTGAGGAAAGCATCATTGAAAACCTTGGTAATGACCACAACGCCGCCGCGGGGGATGGATATCCCAGAAGAACCACACTGACTTCAAAAATATATCACACCAAAGGACAAGAAGGCTCCGTCTGCCTCCGATCATCAGACTGTGCCGCAGGGCTGTGTTGTGCAAGACACTTCTGGTCCAAGATCTGTAAACCTGTCCTTAAAGAAGGTCAGGTGTGCACCAAGCACAAACGGAAAGGCTCCCACGGGCTGGAGATATTCCAGCGCTGTTACTGCGGGGAAGGCCTGGCTTGCAGGATACAGAAAGATCACCATCAAGCCAGCAATTCTTCTAGGCTCCACACCTGCCAGAGACAC   4MMVVCAAAAVRFLAVFTMMALCSLPLLGASATLNSVLINSNAIKNLPPPLGGAGGQPGSAVSVAPGVLYEGGNKYQTLDNYQPYPCAEDEECGSDEYCSSPSRGAAGVGGVQICLACRKRRKRCMRHAMCCPGNYCKNGICMPSDHSHFPRGEIEESIIENLGNDHNAAAGDGYPRRTTLTSKIYHTKGQEGSVCLRSSDCAAGLCCARHFWSKICKPVLKEGQVCTKHKRKGSHGLEIFQRCYCGEGLACRIQKDHHQASNSSRLHTCQRH   5ATGACGGTTGTGCGTGCAGTGGCAGCTGTCCGGTTCTTGGTCGTGCTTACAACGATGGCTCTCTGC RatAGCCTCCCTCCGCTCGGAGTCAGCGCCACTTTGAACTCAGTTCTCATCAATTCCAACGCGATCAAG DKK1AACCTGCCCCCACCGCTGGGTGGTGCTGGGGGGCAGCCGGGCTCTGCTGTCAGCGTGGCGCCCGGAGTCCTCTATGAGGGCGGGAACAAGTACCAGACTCTTGACAACTACCAGCCCTACCCTTGCGCGGAGGATGAGGAGTGCGGCACTGACGAGTACTGCTCCAGTCCCAGCCGCGGGGCAGCCGGCGTGGGAGGTGTACAAATCTGCCTGGCTTGCCGAAAGCGCAGGAAACGCTGCATGAGGCACGCTATGTGCTGCCCCGGGAATTACTGCAAAAACGGAATATGCATGCCCTCTGACCACAGCCATTTACCTCGAGGGGAAATCGAGGAAGGCATCATTGAAAACCTTGGCAATGACCACGGTGCCGGGGATGGATATCCCAGAAGAACCACACTGACTTCAAAAATATATCACACCAAAGGGCAAGAAGGCTCTGTCTGCCTCCGATCATCAGACTGCGCCACAGGGCTGTGTTGTGCAAGACATTTCTGGTCCAAGATCTGTAAACCTGTCCTTAAAGAAGGTCAGGTATGCACCAAGCACAGAAGGAAAGGCTCCCACGGGCTGGAGATATTCCAGCGCTGTTACTGTGGGGAAGGTCTGGCTTGCAGGATACAGAAAGATCACCATCAAACCAGCAATTCTTCCAGGCTCCACACCTGCCAGAGACAC   6MTVVRAVAAVRFLVVLTTMALCSLPPLGVSATLNSVLINSNAIKNLPPPLGGAGGQPGSAVSVAPGVLYEGGNKYQTLDNYQPYPCAEDEECGTDEYCSSPSRGAAGVGGVQICLACRKRRKRCMRHAMCCPGNYCKNGICMPSDHSHLPRGEIEEGIIENLGNDHGAGDGYPRRTTLTSKIYHTKGQEGSVCLRSSDCATGLCCARHFWSKICKPVLKEGQVCTKHRRKGSHGLEIFQRCYCGEGLACRIQKDHHQTSNSSRLHTCQRH   7ATGATGGCTCTGGGCGCAGCAGGAGCTGCCCGGGTCTTGGTCGCGCTGGTAGCGGCGGCTCTTGGC CynoGGCCACCCTCTGCTGGGAGTGAGCGCCACCTTGAACTCGGTTCTCAATTCCAACGCGATCAAGAAC DKK1CTGCCCCCACCGCTGGGCGGCGCTGCGGGGCACCCAGGCTCTGCAGTCAGCGCCGCGCCAGGAATTCTGTACCCGGGCGGGAATAAGTACCAGACCATTGACAACTACCAGCCGTACCCTTGCGCAGAGGATGAGGAGTGCGGCACTGATGAGTACTGCGCTAGTCCCACCCGCGGAGGGGACGCGGGCGTGCAAATCTGTCTCGCCTGCAGGAAGCGCCGAAAACGCTGCATGCGTCACGCTATGTGCTGCCCCGGGAATTACTGCAAAAATGGAATATGTGTGTCTTCTGATCAAAATAATTTCCGAGGGGAAATTGAGGAAACCATTACTGAAAGCTTTGGTAATGATCATAGCACTTTGGATGGGTATTCCAGAAGAACAACATTGTCTTCAAAAATGTATCACAGCAAAGGACAAGAAGGTTCTGTGTGTCTCCGGTCATCAGACTGTGCCACAGGACTGTGTTGTGCTAGACACTTCTGGTCCAAGATCTGTAAACCTGTCCTCAAAGAAGGTCAAGTGTGTACCAAGCATAGAAGAAAAGGCTCTCATGGGCTAGAAATATTCCAGCGTTGTTACTGCGGAGAAGGTCTGTCTTGCCGGATACAGAAAGATCACCATCAAGCCAGTAATTCTTCTAGGCTTCACACTTGTCAGAGACAC   8MMALGAAGAARVLVALVAAALGGHPLLGVSATLNSVLNSNAIKNLPPPLGGAAGHPGSAVSAAPGILYPGGNKYQTIDNYQPYPCAEDEECGTDEYCASPTRGGDAGVQICLACRKRRKRCMRHAMCCPGNYCKNGICVSSDQNNFRGEIEETITESFGNDHSTLDGYSRRTTLSSKMYHSKGQEGSVCLRSSDCATGLCCARHFWSKICKPVLKEGQVCTKHRRKGSHGLEIFQRCYCGEGLSCRIQKDHHQASNSSRLHTCQ RH  9 GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACT2.4.1 TGCCGGGCAAGTCAGGGCATTAGAGATGATTTAGGCTGGTTTCAGCAGAAACCAGGGAAAGCCCCTVk AAGCGCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACAATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTGTCTACAGCATAATAGTTACCCGTGCAGTTTTGGCCAGGGGACCAAGCTGGAGTTCAAA  10DIQMTQSPSSLSASVGDRVTITCRASQGIRDDLGWFQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPCSFGQGTKLEFK  11CAGGTTCAGCTAATGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGC 2.4.1AAGGCTTCTGGTTACACCTTTACCAGCTATGGTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGG VhCTTGAGTGGATGGGATGGATCAGCGCTGACAATGGTCACACAAACTATGCACAGAAACTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGATGGGGAGCTACTAAATTACTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA  12QVQLMQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISADNGHTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDGELLNYYYYYGMDVWGQGTTVTVSS  13GATATTGTGATGACCCAGACTCCACTCTCTCTGTCCGTCATTCCTGGACAGCCGGCCTCCATCTCC2.20.1TGCAAGTCTAGTCAGAGCCTCCTGCATAGTGATGGAAAGACCTATTTGTACTGGTACCTGCAGAGG VkCCAGGCCAGCCTCCACAGCTCCTGATCTATGAAGTTTCCAACCGGTTCTCTGGAGTGCCACATAGGCTCAGTGGCAGCGGGTCAGGGACAGATTTCACACTGAAAATCAGCCGGGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAAGTATACAGGTTCCGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA  14DIVMTQTPLSLSVIPGQPASISCKSSQSLLHSDGKTYLYWYLQRPGQPPQLLIYEVSNRFSGVPHRLSGSGSGTDFTLKISRVEAEDVGVYYCMQSIQVPWTFGQGTKVEIK  15CAGTGTCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTC2.20.1TCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGC VhAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTGATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGATCAATGGGGTGGGAGCCCAGCCGGCCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA  16QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSDKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDQWGGSPAGPWGQGTLVTVSS  17GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCC2.37.1TGCAGGGCCAGTCAGAGTGTTAGCAGCAACTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCT VkCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA  18EIVLTQSPGTLSLSPGERATLSCRASQSVSSNYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPITFGQGTRLEIK  19CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGT2.37.1GCAGCCTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGG VhCTGGAATGGGTGGCAGTTATATCATATGATGGAAGTGATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCTTCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGAATTGGGTATAGCAGCTTCCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA  20QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSDKYYADSVKGRFTFSRDNSKNTLYLQMNSLRAEDTAVYYCARELGIAASFDYWGQGTLVTVSS  21GATATTGTGATGACCCAGTCTCCACTCTCTCTGTCCGTCACCCCTGGACAGCCGGCCTCCATCTCC2.40.1TGCAAGTCTAGTCAGAGCCTCCTGCACAGTGATGGAAAGACCTATTTGTATTGGTATCTGCAGAAG VkCCAGGCCAGCCTCCACAGCTCCTGATCTATGAAGTTTCCAACCGGTTCTCTGGAGTGCCAGATAGGTTCAGTGGCAGCGGGTCAGGGACAGATTTCACACTGAAAATCAGCCGGGTGGAGGCTGAGGATGTTGGGGTCTATTACTGCATGCAAAGTATACAGGTTCCGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA  22DIVMTQSPLSLSVTPGQPASISCKSSQSLLHSDGKTYLYWYLQKPGQPPQLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQSIQVPWTFGQGTKVEIK  23CAGGTGCAACTGGTGGAGTCTGGAGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGT2.40.1GCAGCCTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGG VhCTGGGGTGGGTGGCAGTTATATCATATGATGGAAGTGATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGACCTCGTGGATACAGCTATGCCCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA  24QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLGWVAVISYDGSDKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLVDTAMPWGQGTTVTVSS  25GATATTGTGATGACCCAGACTCCACTCTCTCTGTCCGTCACCCCTGGACAGCCGGCCTCCATCTCC2.41.1TGCAAGTCTAGTCAGAGCCTCCTGCATAGTGATGGAAAGACCTATTTGTATTGGTACCTGCAGAAG VkCCAGGCCAGCCTCCACAGCTCCTGATCTATGAAGTTTCCAACCGGTTCTCTGGAGTGCCAGATAGGTTCAGTGGCAGCGGGTCAGGGACAGATTTCACACTGAAAATCAGCCGGGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAAGTAAACAGCTTCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAA  26DIVMTQTPLSLSVTPGQPASISCKSSQSLLHSDGKTYLYWYLQKPGQPPQLLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQSKQLPFTFGPGTKVDIK  27CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGT2.41.1GCAGCCTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGG VhCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTGATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGGGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGCCGGGTACTCCCTCTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA  28QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSDKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARAGYSLYYYYGMDVWGQGTTVTVSS  29GATATTGTGATGACCCAGACTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCC2.47.1TGCAGGTCTAGTCAGAGCCTCTTGGATAGTGATGATGGAGACACCTATTTGGACTGGTACCTGCAG VkAAGCCAGGGCAGTCTCCACAGCTCCTGATCTATACGCTTTCCTATCGGGCCTCTGGAGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGGAGGCTGAGGATGTTGGAGTTTATTACTGCATGCAACGTATAGAGTTTCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAA  30DIVMTQTPLSLPVTPGEPASISCRSSQSLLDSDDGDTYLDWYLQKPGQSPQLLIYTLSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQRIEFPFTFGPGTKVDIK  31CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCACAGACCCTGTCCCTCACCTGC2.47.1ACTGTCTCTGGTGGCTCCATCAGCAGTGGTGGCTACTACTGGAGCTGGATCCGCCAGCACCCAGGG VhAAGGGCCTGGAGTGGATTGGGGACATCTATTACAGTGGGAGCACCTACTACAACCCGTCCCTCAAGAGTCGAGTTACCATATCAGTAGACACGTCTAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACTGCCGCGGACACGGCCGTGTATTACTGTGCGAGAGATCGGGCTTACGGTGACTACGGGGGAGACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA  32QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGGYYWSWIRQHPGKGLEWIGDIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDRAYGDYGGDYYYGMDVWGQGTTVTVSS  33GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACT5.17.1TGCCAGGCGAGTCAGGACATTAACAACTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCT VkAATCTCCTGATCTACGATGCATCCAATTTGGAAACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGCAGATATTGCAACATATTACTGTCAACAATATGATGATTTCCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA  34DIQMTQSPSSLSASVGDRVTITCQASQDINNYLNWYQQKPGKAPNLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPADIATYYCQQYDDFPLTFGGGTKVEIK  35CAGGTGCAACTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGC5.17.1ACTGTCTCTGGTGGCTCCATCAGTAGTTACTACTGGAGCTGGATCCGGCAGCCCCCAGGGAAGGGA VhCTGGAATGGATTGGGTATATCTATTACAGTGGGAACACCAATTACAACCCCTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGGTCTGTGACCGCTGCGGACACGGCCGTATATTACTGTGCGAGGTATAACTGGAACAACGACCTCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA  36QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYIYYSGNTNYNPSLKSRVTISVDTSKNQFSLKLRSVTAADTAVYYCARYNWNNDLFDYWGQGTLVTVSS  37GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACT5.23.1TGCCAGGCGAGTCAGGACATTAGCAACTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCT VkAAGCTCCTGATCTACGATGCATCCAATTTGGAAACTGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATATTACTGTCAACAATATGATAATCTCCCTCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA  38DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPLTFGGGTKVEIK  39CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGT5.23.1GCAGCGTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGG VhCTGGAGTGGGTGGCAGTTATATGGTGTGATGGAAGTAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAGTTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGGGGGCTATGGTTCGGGGAGTTATGAGGACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA  40QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWCDGSNKYYADSVKGRFTISRDSSKNTLYLQMNSLRAEDTAVYYCARGGYGSGSYEDYYYGMDVWGQGTTVTVSS  41GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACT5.25.1TGCCAGGCGAGTCAGGACATTAGTAAGGATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCT VkAGGCTCCTGATCTACGATGCATCCAATTTGGAAACGGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATTTTACTGTCAACAGTATGATCATCTCCCGATCGCCTTCGGCCAAGGGACACGACTGGAGATTAAA  42DIQMTQSPSSLSASVGDRVTITCQASQDISKDLNWYQQKPGKAPRLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATFYCQQYDHLPIAFGQGTRLEIK  43CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGC5.25.1AAGGCTTCTGGATACACCTTCACCAGTTATGATATCAACTGGGTGCGACAGGCCACTGGCCAAGGG VhCTTGAGTGGATGGGATGGATGGACCCTAACAGTGGTAACACAGGCTATGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGAACACCTCCATAAGCACAGCCTTCATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAACGGACTACTTCTACTTCGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA  44QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDINWVRQATGQGLEWMGWMDPNSGNTGYAQKFQGRVTMTRNTSISTAFMELSSLRSEDTAVYYCARTDYFYFGMDVWGQGTTVTVSS  45GACATCCAGGTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACT5.31.1TGCCAGGCGAGTCAGGACATTAGCAACTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCT VkAAGTTCCTGATCTACGATGCATCCAATTTGGAAGCAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATATTACTGTCAACAGTATGATAATCTCCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA  46DIQVTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKFLIYDASNLEAGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPLTFGGGTKVEIK  47CAGGTGCAGTTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGT5.31.1GCAGCGTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGG VhCTGGAGTGGGTGGCAGTTATATGGTATGATGGAAGAAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGGGGGGGGAGCAGTGGCTGATTACAACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA  48QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGRNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGGAVADYNYYYGMDVWGQGTTVTVSS  49GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACT5.32.1TGCCAGGCGAGTCAGGACATTAGCAAGGATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCT VkAAGCTCCTGATCTACGATGCATCCAATTTGGAAACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATATTACTGTCAACAGTATGATGATCTCCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA  50DIQMTQSPSSLSASVGDRVTITCQASQDISKDLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDDLPITFGQGTRLEIK  51CAGGTGCAGCTGGTACAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGC5.32.1AAGGCCTCTGGATTCACCTTCACCAGTTATGATATCAGCTGGGTGCGACAGGCCACTGGACTAGGG VhCTTGAGTGGATGGGATGGATGAACCCTAGCAGTGGTTACACAGGCTATGCACAGAACTTCCAGGGCAGAGTCACCATGACCTGGAACACCTCCATAAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAACGGACTACTACTACTACGGTATGGACGTCTGGGGCCGAGGGACCACGGTCACCGTCTCCTCA  52QVQLVQSGAEVKKPGASVKVSCKASGFTFTSYDISWVRQATGLGLEWMGWMNPSSGYTGYAQNFQGRVTMTWNTSISTVYMELSSLRSEDTAVYYCARTDYYYYGMDVWGRGTTVTVSS  53GACATCCGGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACT5.40.1TGCCAGGCGAGTCAGGACATTAGCAACTATTTAAATTGGTATCAGCAGGAACCAGGGAAAGCCCCT VkAAGCTCCTGATCTACGATGCATCCAATTTGGAAACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATATTACTGTCAACAGTATGATAATTTCCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA  54DIRLTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQEPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNFPLTFGGGTKVEIK  55CAGGTGCTACTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGC5.40.1ACTGTCTCTGGTGGCTCCATCAGTAGTTACTACTGGAGCTGGATCCGGCAGACCCCAGGGAAGGGA VhCTGGAGTGGATTGGGTATGTCTATTACAGTGGGAGCACCAGCTACAACCCCTCCCTCAAGAGTCGAGTCACCATATCAATGTACACGTCCAAGACCGAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCTGCGGACACGGCCGTGTATTACTGTGCGAGGTATAACTGGAACAACGACCTCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA  56QVLLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQTPGKGLEWIGYVYYSGSTSYNPSLKSRVTISMYTSKTEFSLKLSSVTAADTAVYYCARYNWNNDLFDYWGQGTLVTVSS  57TCCTATGTGTTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGACAGACGGCCAGGATTACCTGT5.65.1GGGGGAAACAACATTGGAAGTAAAAGTGTGCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTG VlCTGGTCGTCTATGATGATAGCGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAAGCCGGGGATGAGGCCGACTATTACTGTCAGGTGTTGGATAGTAGTAGTGATCATGTGATATTCGGCGGAGGGACCAAGCTGACCGTCCTA  58SYVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVLDSSSDHVIFGGGTKLTVL  59TCCTATGTGTTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGACAGACGGCCAGGATTACCTGT5.65.1GGGGGAAACAACATTGGAAGTAAAAGTGTGCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTG VhCTGGTCGTCTATGATGATAGCGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAAGCCGGGGATGAGGCCGACTATTACTGTCAGGTGTTGGATAGTAGTAGTGATCATGTGATATTCGGCGGAGGGACCAAGCTGACCGTCCTA  60EVQLLESGGGLVQPGGSLRLSCAASGFTSGNYAMSWVRQAPGKGLEWVSAISGGGGTTYYADSVEGRFTISRDNSKNTLYLQLNSLRAEDTAVYYCAKEFGELEPRFDYWGQGTLVTVSS  61TCCTATGTGCTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGACAGACGGCCAGGATTACCTGT5.76.1GGGGGAAACAACATTGGAAGTGAAAGTGTGCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTG VlCTGGTCGTCTATGATGATAGCGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAAGCCGGGGATGAGGCCGACTATTACTGTCAGGTGTGGGATAGTAGTAATGATCATGTGGTTTTCGGCGGAGGGACCAAGCTGACCGTCCTA  62SYVLTQPPSVSVAPGQTARITCGGNNIGSESVHWYQQKPGQAPVLVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSNDHVVFGGGTKLTVL  63CAGCTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGC5.76.1ACTGTCTCTGGTGGCTCCATCAGCAGTAGTAATTACTACTGGGGCTGGATCCGCCAGCCCCCGGGG VhAAGGGGCTGGAGTGGATTGGGACTATCTATTATAGTGGGAGCACCTACTACACCCCGTCCCTCAAGAGTCGAGTCACCATATCCGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTCTATTACTGTGCGAGAGAGAGGGCGATAGCAGTGGCTGCTATAGTCTTCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA  64QLQLQESGPGLVKPSETLSLTCTVSGGSISSSNYYWGWIRQPPGKGLEWIGTIYYSGSTYYTPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARERAIAVAAIVFFDYWGQGTLVTVSS  65TCCTATGTGCTGACTCAGTCACCCTCGGTGTCAGTGGCCCCAGGACAGACGGCCAGGATTACCTGT5.77.1GGGGGAAACAACATTGGAAGTAAAAGTGTGCACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTG VlCTGGTCGTCTATGATGATAGCGACCGGCCCTCAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAAGCCGGGGATGAGGCCGACTACTACTGTCAGGTGTGGGATAGTAGTAGTGATCATTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA  66QSPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHWVFGGGTKLTVL  67CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGC5.77.1AAGGCTTCTGGATACACCTTCACCAGTTATGATATCAACTGGGTGCGACAGGCCACTGGACAAGGG VhCTTGAGTGGATGGGATGGATGAATCTTAACAGTGATAACACAGGCTATGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGAACACCTCCATAAGCACTGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGTATAGCAGCTCGTCGCGACTACAACTACTACGGTATGGACGTCTGGGGCCAAGGGACCAAGGTCACCGTCTCCTCA  68QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDINWVRQATGQGLEWMGWMNLNSDNTGYAQKFQGRVTMTRNTSISTAYMELSSLRSEDTAVYYCASIAARRDYNYYGMDVWGQGTKVTVSS  69GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTGTGTCTCCAGGGGAAAGAGCCACCCTCTCC5.78.1TGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTATCAGCAGAAACCTGGCCAGGCT VkCCCAGGCTCCTCATCTATGGTGCATCCGGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCATTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCATTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA  70EIVLTQSPGTLSVSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASGRATGIPDRFSGSGSGTDFILTISRLEPEDFAVYYCQQYGSSFTFGGGTKVEIK  71CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGT5.78.1GCAGCCTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGG VhCTGGAGTGGGTGGCAGTTATATTATATGATGGAAGTGATAATTACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGAAGGGATAGCAGTGGCTGGGGACTACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA  72QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVILYDGSDNYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREGIAVAGDYYYYGMDVWGQGTTVTVSS  73CAGTCAGTGCTGACGCAGCCGCCCTCAGTGTCTGGGGCCCCAGGGCAGAGGGTCACCATCTCCTGC5.80.1ACTGGGAGCAGCTCCAACATCGGGGCAGATTATGATGTACACTGGTACCAGCAGCTTCCAGGAACA VlGCCCCCAAACTCCTCATCTATGATTACAGCAATCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCACTGGGCTCCAGGCTGAGGATGAGGCTGATTATTACTGCCAGTCCTATGACAACAGCCTGAGTGGTTATGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTA  74QSVLTQPPSVSGAPGQRVTISCTGSSSNIGADYDVHWYQQLPGTAPKLLIYDYSNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDNSLSGYVVFGGGTKLTVL  75GAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGT5.80.1AAGGGTTCTGGATACAGCTTTACCAGCTACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGC VhCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAGCAGCCTGACGGCCTCGGACACCGCCATGTATTACTGTGCGAGACAGGGAGAGAGCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA  76EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLTASDTAMYYCARQGESFDYWGQGTLVTVSS  77CAGTCTGTGCTGACGCAGCCGCCCTCAGTGTCTGGGGCCCCAGGGCAGAGGGTCACCATCTCCTGC5.85.1ACTGGGAGCAGCTCCAACATCGGGGCAGGTTATGATGTACACTGGTACCAGCAGCTTCCAAGAACA VlGCCCCCAAACTCCTCATCTATGGTAACAGCAATCGGCCCTCAGGGGTCCCTGACCGATTCTCTGACTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCACTGGCCTCCAGGCTGAGGATGAGGCTGATTATTACTGCCAGTCCTATGACAGCAGCCTGAGTGTGATATTCGGCGGAGGGACCAAGCTGACCGTCCTA  78QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPRTAPKLLIYGNSNRPSGVPDRFSDSKSGTSASLAITGLQAEDEADYYCQSYDSSLSVIFGGGTKLTVL  79GAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGT5.85.1AAGGTTTCTGGATACAGCTTTACCACCTACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGC VhCTGGACTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGAGACAAGGTATAGCGTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA  80EVQLVQSGAEVKKPGESLKISCKVSGYSFTTYWIGWVRQMPGKGLDWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARQGIAFDYWGQGTLVTVSS  81GATATTGTGATGACCCAGACTCCACTCTCTCTGTCCGTCACCCCTGGACAGCCGGCCTCCATCTCC6.37.5TGCAAGTCTGGTCAGAGCCTCCTGCATAGTGATGGAAAGACCTATTTGTATTGGTACCTGCAGAAG VkCCAGGCCAGCCTCCACAGTTCCTGATCTATGAAGTTTCCAACCGGTTCTCTAGAGTGCCAGATAGGTTCAGTGGCAGCGGGTCAGGGACAGATTTCACACTGAGAATCAGCCGGGTGGAGGCTGAGGATGTTGGAATTTATTACTGCATGCAAAGTATACAGCTTCCGTGGACGTTCGGCCAAGGGACCCAGGTGGAAATCAAA  82DIVMTQTPLSLSVTPGQPASISCKSGQSLLHSDGKTYLYWYLQKPGQPPQFLIYEVSNRFSRVPDRFSGSGSGTDFTLRISRVEAEDVGIYYCMQSIQLPWTFGQGTQVEIK  83CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGT6.37.5GCAGCCTCTGGATTCACCTTCAGTGGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGG VhCTGGAGTGGGTGGCAGTTATATCATATGATGGAAATGATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAGAGAGCTACGGGTCCTCTGGGGCCAGGGAACCCTGGTCACCGTCTCTAGT  84QVQLVESGGGVVQPGRSLRLSCAASGFTFSGYGMHWVRQAPGKGLEWVAVISYDGNDKYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARELRVLWGQGTLVTVSS  85GATATTGTGATGACCCAGACTCCACTCTCTCTGTCCGTCACCCCTGGACAGCCGGCCTCCATCTCC6.116.6TGCAAGTCTGGTCAGAGCCTCCTGCATAATGATGGAAAGACCTATTTGTATTGGTACCTGCAGAAG VkCCAGGCCAGCCTCCACAGTTCCTGATCTATGAAGTTTCCAACCGGTTCTCTAGAGTGCCAGATAGGTTCAGTGGCAGCGGGTCAGGGACAGATTTCACACTGAAAATCAGCCGGGTGGAGGCTGAGGATGTTGGAATTTATTACTGCATGCAAAGTATACAGCTTCCGTGGACGTTCGGCCAAGGGACCCAGGTGGAAATCAAA  86DIVMTQTPLSLSVTPGQPASISCKSGQSLLHNDGKTYLYWYLQKPGQPPQFLIYEVSNRFSRVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQSIQLPWTFGQGTQVEIK  87CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGT6.116.6GCAGCCTCTGGATTCACCTTCAGTGGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGG VhCTGGAGTGGGTGGCAGTTATATCATATGATGGAAATGATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTTTATTACTGTGCGAGAGAGCTACGGGTCCTCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA  88QVQLVESGGGVVQPGRSLRLSCAASGFTFSGYGMHWVRQAPGKGLEWVAVISYDGNDKYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARELRVLWGQGTLVTVSS  89GATATTGTGATGACCCAGACTCCACTCTCTCTGTCCGTCACCCCTAGACAGCCGGCCTCCATCTCC6.139.5TGCAAGTCTAGTCAGAGCCTCCTGCATAGTGATGGAAAGACCTATTTGTATTGGTACCTGCAGAAG VkCCAGGCCAGCCTCCACAGTTCCTGATCTATGAAGTTTCCAACCGGTTCTCTGGAGTGCCAGATAGGTTCAGTGGCAGCGGGTCAGGGACAGATTTCACACTGAAAATCAGCCGGGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAAGTATACAGCTTCCGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA  90DIVMTQTPLSLSVTPRQPASISCKSSQSLLHSDGKTYLYWYLQKPGQPPQFLIYEVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQSIQLPWTFGQGTKVEIK  91CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGT6.139.5GCAGCCTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGG VhCTGGAGTGGGTGGCAGTTATATCATATGATGGAGGTGATCAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAACTGAGGACACGGCTGAGTATTACTGTGCGAGAGAGCTCCGGGTCCTCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA  92QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGGDQYYADSVKGRFTISRDNSKNTLYLQMNSLRTEDTAEYYCARELRVLWGQGTLVTVSS  93TCCTATGTGCTGACTCAGCCACCCTCGGTGTCAGTGGCCCCAGGACAGACGGCCAGGATTACCTGT6.147.4GGGGGAAACAACATTGGAAGTAAAAGTGTACACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTG VlCTGGTCGTCTATGATGATAGTGACCGGCCCTCAGAGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAAGCCGGGGATGAGGCCGACTATTACTGTCAGGTGTGGGATAGTAGTAGTGATCATGTGGTATTCGGCGGAGGGACCAGGCTGACCGTCCTA  94YVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDDSDRPSEIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHVVFGGGTRLTVL  95CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGT6.147.4GCAGCGTCTGGATTCACCTTCAGTCGCTATGACATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGG VhCTGGAGTGGGTGGCAATTATATTCTATGATGGCAGCAATAAATACTATGCAGACCCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCAAAGAACACACTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGACTCTAGCAGCAGCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA  96QVQLVESGGGVVQPGRSLRLSCAASGFTFSRYDMHWVRQAPGKGLEWVAIIFYDGSNKYYADPVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATLAAAFDYWGQGTLVTVSS  97 RASQGIRDDLG 2.4.1 98 AASSLQS  99 LQHNSYPCS 100 SYGIS 101 WISADNGHTNYAQKLQG 102DGELLNYYYYYGMDV 103 KSSQSLLHSDGKTYLY 2.20.1 104 EVSNRFS 105 MQSIQVPWT106 SYGMH 107 VISYDGSDKYYADSVKG 108 DQWGGSPAGP 109 RASQSVSSNYLA 2.37.1110 GASSRAT 111 QQYGSSPIT 112 SYGMH 113 VISYDGSDKYYADSVKG 114 ELGIAASFDY115 KSSQSLLHSDGKTYLY 2.40.1 116 EVSNRFS 117 MQSIQVPWT 118 SYGMH 119VISYDGSDKYYADSVKG 120 DLVDTAMP 121 KSSQSLLHSDGKTYLY 2.41.1 122 EVSNRFS123 MQSKQLPFT 124 SYGMH 125 VISYDGSDKYYADSVKG 126 AGYSLYYYYGMDV 127RSSQSLLDSDDGDTYLD 2.47.1 128 TLSYRAS 129 MQRIEFPMQRIEFP 130 SGGYYWS 131DIYYSGSTYYNPSLKS 132 DRAYGDYGGDYYYGMDV 133 QASQDINNYLN 5.17.1 134DASNLET 135 QQYDDFPLT 136 SYYWS 137 YIYYSGNTNYNPSLKS 138 YNWNNDLFDY 139QASQDISNYLN 5.23.1 140 DASNLET 141 QQYDNLPLT 142 SYGMH 143VIWCDGSNKYYADSVKG 144 GGYGSGSYEDYYYGMDV 145 QASQDISKDLN 5.25.1 146DASNLET 147 QQYDNLPLT 148 SYGMH 149 WMDPNSGNTGYAQKFQG 150 TDYFYFGMDV 151QASQDISNYLN 5.31.1 152 DASNLEA 153 QQYDNLPLT 154 SYGMH 155VIWYDGRNKYYADSVKG 156 GGGAVADYNYYYGMDV 157 QASQDISKDLN 5.32.1 158DASNLET 159 QQYDDLPIT 160 SYDIS 161 WMNPSSGYTGYAQNFQG 162 TDYYYYGMDV 163QASQDISNYLN 5.40.1 164 DASNLET 165 QQYDNFPLT 166 SYYWS 167YVYYSGSTSYNPSLKS 168 YNWNNDLFDY 5.65.1 169 GGNNIGSKSVH 170 DDSDRPS 171QVLDSSSDHVI 172 NYAMS 173 AISGGGGTTYYADSVEG 174 EFGELEPRFDY 175GGNNIGSESVH 5.76.1 176 DDSDRPS 177 QVWDSSNDHVV 178 SSNYYWG 179TIYYSGSTYYTPSLKS 180 ERAIAVAAIVFFDY 181 GGNNIGSKSVH 5.77.1 182 DDSDRPS183 QVWDSSSDHWV 184 SYDIN 185 WMNLNSDNTGYAQKFQG 186 IAARRDYNYYGMDV 187RASQSVSSSYLA 5.78.1 188 GASGRAT 189 QQYGSSFT 190 SYGMH 191VILYDGSDNYYADSVKG 192 EGIAVAGDYYYYGMDV 193 TGSSSNIGADYDVH 5.80.1 194DYSNRPS 195 QSYDNSLSGYVV 196 SYWIG 197 IIYPGDSDTRYSPSFQG 198 QGESFDY 199TGSSSNIGAGYDVH 5.85.1 200 GNSNRPS 201 QSYDSSLSVI 202 TYWIG 203IIYPGDSDTRYSPSFQG 204 QGIAFDY 205 KSGQSLLHSDGKTYLY 6.37.5 206 EVSNRFS207 MQSIQLPWT 208 GYGMH 209 VISYDGNDKYYADSVKG 210 ELRVL 211KSGQSLLHNDGKTYLY 6.116.6 212 EVSNRFS 213 MQSIQLPWT 214 GYGMH 215VISYDGNDKYYADSVKG 216 ELRVL 217 AASGFTFSRYDMH 6.139.5 218 IFYDGSNKYYAD219 ATLAAAFDY 220 SYGMH 221 VISYDGGDQYYADSVKG 222 ELRVL 223 GGNNIGSKSVH6.147.4 224 DDSDRPS 225 QVWDSSSDHVV 226 RYDMH 227 IIFYDGSNKYYADPVKG 228LAAAFDY 229CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACT kappaGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGAT lightAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTAC chainAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAG 230RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 231GGTCAGCCCAAGGCCAACCCCACTGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTCCAAGCCAAClambdaAAGGCCACACTAGTGTGTCTGATCAGTGACTTCTACCCGGGAGCTGTGACAGTGGCCTGGAAGGCA C1GATGGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCAAACCCTCCAAACAGAGCAACAACAAGTAC lightGCGGCCAGCAGCTACCTGAGCCTGACGCCCGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAG chainGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCA 232GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 233GGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAAClambdaAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCA C2GATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTAC lightGCGGCCAGCAGCTATCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAG chainGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCA 234GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 235GGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCACCCTCCTCTGAGGAGCTTCAAGCCAAClambdaAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCA C3GATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTAC lightGCGGCCAGCAGCTACCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAAAAGCTACAGCTGCCAG chainGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCA 236GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTVAPTECS 237GGTCAGCCCAAGGCTGCCCCATCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAAClambdaAAGGCCACACTGGTGTGCCTGATCAGTGACTTCTACCCGGGAGCTGTGAAAGTGGCCTGGAAGGCA C6GATGGCAGCCCCGTCAACACGGGAGTGGAGACCACCACACCCTCCAAACAGAGCAACAACAAGTAC lightGCGGCCAGCAGCTACCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAG chainGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTGCAGAATGTGCA 238GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVKVAWKADGSPVNTGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPAECA 239GGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCACCCTCCTCTGAGGAGCTTCAAGCCAAClambdaAAGGCCACACTGGTGTGTCTCGTAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCA C7GATGGCAGCCCCGTCAAGGTGGGAGTGGAGACCACCAAACCCTCCAAACAAAGCAACAACAAGTAT lightGCGGCCAGCAGCTACCTGAGCCTGACGCCCGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCGG chainGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTGCAGAATGCTCT 240GQPKAAPSVTLFPPSSEELQANKATLVCLVSDFYPGAVTVAWKADGSPVKVGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCRVTHEGSTVEKTVAPAECS 241GCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACA IgG2GCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGC heavyGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGC chainAGCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 242ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 243GCCAGCACCAAGGGGCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACA IgG4GCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGC heavyGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGC chainAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCATCATGCCCAGCACCTGAGTTCGAGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAA 244ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

One of skill in the art will appreciate the distinction betweensequences shown in Table 1 that encompass variable regions of both theheavy and light chains compared to sequences of the full lengthantibody, which additional comprise constant regions. Variable domainscan be combined with appropriate constant domains using standardtechnology well known in the art. Each of the light chains listed inTable 1 can be combined with any of the heavy chains shown in Table 1(e.g., polypeptides depicted in SEQ ID NOs: 242 or 244) to form anantibody. In some instances, the antibodies include at least one heavychain and one light chain from those listed in Table 1. In otherinstances, the antibodies contain two identical light chains and twoidentical heavy chains. As an example, an antibody or immunologicallyfunctional fragment may include two L1 light chains and two H1 heavychains, or two L2 light chains and two H3 heavy chains, or two L2 lightchains and two H4 heavy chains or two L2 and two H5 heavy chains andother similar combinations of pairs of light chains and pairs of heavychains as listed in Table 1.

Other antibodies that are provided are variants of antibodies formed bycombination of the heavy and light chains shown in Table 1 and compriselight and/or heavy chains that each have at least 70%, 75%, 80%, 85%,90%, 95%, 97% or 99% identity to the amino acid sequences of thesechains. In some instances, such antibodies include at least one heavychain and one light chain, whereas in other instances such variant formscontain two identical light chains and two identical heavy chains.

Certain antibodies comprise a light chain variable domain comprising asequence of amino acids that differs from the sequence of a light chainvariable domain described herein at only 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14 or 15 amino acid residues, wherein each such sequencedifference is independently either a deletion, insertion or substitutionof one amino acid. The light chain variable region in some antibodiescomprises a sequence of amino acids that has at least 70%, 75%, 80%,85%, 90%, 95%, 97% or 99% sequence identity to the amino acid sequencesof the light chain variable regions in Table 1

Some antibodies that are provided comprise a heavy chain variable domaincomprising a sequence of amino acids that differs from the sequence of aheavy chain variable domain in Table 1 at only 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14 or 15 amino acid residues, wherein each suchsequence difference is independently either a deletion, insertion orsubstitution of one amino acid. The heavy chain variable region in someantibodies comprises a sequence of amino acids that has at least 70%,75%, 80%, 85%, 90%, 95%, 97% or 99% sequence identity to the amino acidsequences of the heavy chain variable region shown in Table 1. Stillother antibodies or immunologically functional fragments include variantforms of a variant light chain and a variant heavy chain as justdescribed.

Complementarity determining regions (CDRs) and framework regions (FR) ofa given antibody may be identified using the system described by Kabatet al. in Sequences of Proteins of Immunological Interest, 5th Ed., USDept. of Health and Human Services, PHS, NIH, NIH Publication no.91-3242, 1991.

The antibodies and immunological functional fragments that are providedcan include one, two, three, four, five or all six of the CDRs listedabove. Some antibodies or fragments include both the light chain CDR3and the heavy chain CDR3. Certain antibodies have variant forms of theCDRs, with one or more (i.e., 2, 3, 4, 5 or 6) of the CDRs each havingat least 80%, 85%, 90% or 95% sequence identity to a CDR sequence. Forexample, the antibody or fragment can include both a light chain CDR3and a heavy chain CDR3 that each have at least 80%, 85%, 90% or 95%sequence identity to the light chain CDR3 sequence and the heavy chainCDR3, respectively. The CDR sequences of some of the antibodies that areprovided may also differ from the CDR sequences in Table 1 such that theamino acid sequence for any given CDR differs from the sequence listedin Table 1 by no more than 1, 2, 3, 4 or 5 amino acid residues.Differences from the listed sequences usually are conservativesubstitutions (see below).

Polypeptides comprising one or more of the light or heavy chain CDRs maybe produced by using a suitable vector to express the polypeptides in asuitable host cell as described in greater detail below. The heavy andlight chain variable regions and the CDRs that are disclosed in Table 1can be used to prepare any of the various types of immunologicallyfunctional fragments that are known in the art including, but notlimited to, domain antibodies, Fab fragments, Fab′ fragments, F(ab′)₂fragments, Fv fragments, single-chain antibodies and scFvs.

When an antibody is said to bind an epitope within specified residues,such as DKK1, for example, what is meant is that the antibodyspecifically binds to a polypeptide consisting of the specified residues(e.g., a specified segment of DKK1). Such an antibody does notnecessarily contact every residue within DKK1. Nor does every singleamino acid substitution or deletion within DKK1 necessarilysignificantly affect binding affinity. Exact epitope specificity of anantibody can be determined in variety of ways. One approach, forexample, involves testing a collection of overlapping peptides of about15 amino acids spanning the sequence of DKK1 and differing in incrementsof a small number of amino acids (e.g., 3 amino acids). The peptides areimmobilized within the wells of a microtiter dish. Immobilization can beeffected by biotinylating one terminus of the peptides. Optionally,different samples of the same peptide can be biotinylated at the N and Cterminus and immobilized in separate wells for purposes of comparison.This is useful for identifying end-specific antibodies. Optionally,additional peptides can be included terminating at a particular aminoacid of interest. This approach is useful for identifying end-specificantibodies to internal fragments of DKK1. An antibody or immunologicallyfunctional fragment is screened for specific binding to each of thevarious peptides. The epitope is defined as occurring with a segment ofamino acids that is common to all peptides to which the antibody showsspecific binding.

Antibodies and functional fragments thereof that bind to aconformational epitope that is located in the carboxy-terminal portionof DKK1 (see Table 1) are also provided. The carboxy-terminus of DKK1contains several cysteine residues that form a cluster of disulfidebonds which create several loops. The invention provides antibodies thatbind to two of these loops, thereby neutralizing the ability of DKK1 tosuppress Wnt activity. Exemplary antibodies capable of binding to theaforementioned conformational epitope are the monoclonal antibodies11H10 and 1F11, each of which comprises a light chain and a heavy chain.These antibodies are described in detail in U.S. Pat. No. 7,709,611.

The epitope comprising these two loops is formed by disulfide bondsbetween cysteine residues 220 and 237 of SEQ ID NO: 2 and betweencysteine residues 245 and 263 of SEQ ID NO:2. The body of the two loopsthat form the epitope thus includes amino acids 221-236 and 246-262 ofSEQ ID NO: 2. Segments within this loop that are involved in bindinginclude amino acids 221-229 of SEQ ID NO:2 and amino acids 246-253 ofSEQ ID NO: 2. Thus, certain antibodies and fragments that are providedherein specifically bind to the foregoing region(s). Some of theantibodies and fragments, for instance, bind to a peptide comprising orconsisting of amino acids 221 to 262 of SEQ ID NO: 2.

In one aspect of the invention, peptides comprising or consisting ofamino acids 221-229 and/or 246-253 of SEQ ID NO: 2 are provided. Otherpeptides comprise or consist of amino acids 221-236 and/or 246-262 ofSEQ ID NO: 2. Still other peptides that are provided comprise or consistof the region from 221 to 262 of SEQ ID NO: 2 or amino acids 221-253 ofSEQ ID NO:2. Such peptides are shorter than the full-length proteinsequence of a native DKK1 (e.g., the peptides may include one or more ofthe forgoing regions and be 8, 9, 10, 11, 12, 13, 14, 15, 20, 21, 22,23, 24, 25, 30, 40, 50, 75, 100, 150, or 200 amino acids in length).These peptides may be fused to another peptide to increaseimmunogenicity and thus be in the form of a fusion protein.

Antibodies and immunologically functional fragments thereof that competewith one the exemplified antibodies or functional fragments for specificbinding to DKK1 are also provided. Such antibodies and fragments mayalso bind to the same epitope as one of the exemplified antibodies.Antibodies and fragments that compete with or bind to the same epitopeas the exemplified antibody or fragment are expected to show similarfunctional properties. The exemplified antibodies and fragment includethose described above, including those with the heavy and light chains,variable region domains and CDRs listed in Table 1. Competing antibodiesor immunologically functional fragments can include those that bind tothe epitope described in the section on antibodies and epitopes above.

As a specific example, some competing antibodies or fragments includethose that specifically bind a DKK1 protein consisting of amino acids 32to 266 of SEQ ID NO: 2 and can prevent or reduce the binding to humanDKK1 of an antibody that consists of two identical heavy chains and twoidentical light chains. Other competing antibodies prevent or reduce thebinding to human DKK1 of an antibody that consists of two identicalheavy chains and two identical light chains such as those listed inTable 1.

The antibodies that are provided include monoclonal antibodies that bindto DKK1. Monoclonal antibodies may be produced using any technique knownin the art, e.g., by immortalizing spleen cells harvested from thetransgenic animal after completion of the immunization schedule. Thespleen cells can be immortalized using any technique known in the art,e.g., by fusing them with myeloma cells to produce hybridomas. Myelomacells for use in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render them incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas). Examples of suitable cell lines for use in mouse fusionsinclude Sp-20, P3-X63/Ag8, P3-X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO,NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XXO Bul; examples of celllines used in rat fusions include R210.RCY3, Y3-Ag 1.2.3, IR983F and4B210. Other cell lines useful for cell fusions are U-266, GM1500-GRG2,LICR-LON-HMy2 and UC729-6.

In some instances, a hybridoma cell line is produced by immunizing ananimal (e.g., a transgenic animal having human immunoglobulin sequences)with a DKK1 immunogen; harvesting spleen cells from the immunizedanimal; fusing the harvested spleen cells to a myeloma cell line,thereby generating hybridoma cells; establishing hybridoma cell linesfrom the hybridoma cells, and identifying a hybridoma cell line thatproduces an antibody that binds a DKK1 polypeptide. Such hybridoma celllines, and anti-DKK1 monoclonal antibodies produced by them, areencompassed by the present invention.

Monoclonal antibodies secreted by a hybridoma cell line can be purifiedusing any useful technique known in the antibody arts. Hybridomas ormAbs may be further screened to identify mAbs with particularproperties, such as the ability to block a Wnt induced activity.Examples of such screens are provided in the examples below.

Chimeric and humanized antibodies based upon the foregoing sequences arealso provided. Monoclonal antibodies for use as therapeutic agents maybe modified in various ways prior to use. One example is a “chimeric”antibody, which is an antibody composed of protein segments fromdifferent antibodies that are covalently joined to produce functionalimmunoglobulin light or heavy chains or immunologically functionalportions thereof. Generally, a portion of the heavy chain and/or lightchain is identical with or homologous to a corresponding sequence inantibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is/are identical with or homologous to a corresponding sequencein antibodies derived from another species or belonging to anotherantibody class or subclass. For methods relating to chimeric antibodies,see, for example, U.S. Pat. No. 4,816,567; and Morrison et al., Proc.Natl. Acad. Sci. USA 81:6851-6855 (1985). CDR grafting is described, forexample, in U.S. Pat. Nos. 6,180,370, 5,693,762, 5,693,761, 5,585,089,and 5,530,101.

Generally, the goal of making a chimeric antibody is to create a chimerain which the number of amino acids from the intended patient species ismaximized. One example is the “CDR-grafted” antibody, in which theantibody comprises one or more complementarity determining regions(CDRs) from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the antibody chain(s) is/areidentical with or homologous to a corresponding sequence in antibodiesderived from another species or belonging to another antibody class orsubclass. For use in humans, the V region or selected CDRs from a rodentantibody often are grafted into a human antibody, replacing thenaturally-occurring V regions or CDRs of the human antibody.

One useful type of chimeric antibody is a “humanized” antibody.Generally, a humanized antibody is produced from a monoclonal antibodyraised initially in a non-human animal. Certain amino acid residues inthis monoclonal antibody, typically from non-antigen recognizingportions of the antibody, are modified to be homologous to correspondingresidues in a human antibody of corresponding isotype. Humanization canbe performed, for example, using various methods by substituting atleast a portion of a rodent variable region for the correspondingregions of a human antibody (see, e.g., U.S. Pat. Nos. 5,585,089, and5,693,762; Jones et al., 1986, Nature 321:522-25; Riechmann et al.,1988, Nature 332:323-27; Verhoeyen et al., 1988, Science 239:1534-36).In certain embodiments, constant regions from species other than humancan be used along with the human variable region(s) to produce hybridantibodies.

Fully human antibodies are also provided. Methods are available formaking fully human antibodies specific for a given antigen withoutexposing human beings to the antigen (“fully human antibodies”). Onemeans for implementing the production of fully human antibodies is the“humanization” of the mouse humoral immune system. Introduction of humanimmunoglobulin (Ig) loci into mice in which the endogenous Ig genes havebeen inactivated is one means of producing fully human monoclonalantibodies (MAbs) in mouse, an animal that can be immunized with anydesirable antigen. Using fully human antibodies can minimize theimmunogenic and allergic responses that can sometimes be caused byadministering mouse or mouse-derivatized Mabs to humans as therapeuticagents.

Fully human antibodies can be produced by immunizing transgenic animals(usually mice) that are capable of producing a repertoire of humanantibodies in the absence of endogenous immunoglobulin production.Antigens for this purpose typically have six or more contiguous aminoacids, and optionally are conjugated to a carrier, such as a hapten.See, for example, Jakobovits et al., 1993, Proc. Natl. Acad. Sci. USA90:2551-2555; Jakobovits et al., 1993, Nature 362:255-258; andBruggermann et al., 1993, Year in Immunol. 7:33. In one example of sucha method, transgenic animals are produced by incapacitating theendogenous mouse immunoglobulin loci encoding the mouse heavy and lightimmunoglobulin chains therein, and inserting into the mouse genome largefragments of human genome DNA containing loci that encode human heavyand light chain proteins. Partially modified animals, which have lessthan the full complement of human immunoglobulin loci, are thencross-bred to obtain an animal having all of the desired immune systemmodifications. When administered an immunogen, these transgenic animalsproduce antibodies that are immunospecific for the immunogen but havehuman rather than murine amino acid sequences, including the variableregions. For further details of such methods, see, for example,WO96/33735 and WO94/02602. Additional methods relating to transgenicmice for making human antibodies are described in U.S. Pat. Nos.5,545,807; 6,713,610; 6,673,986; 6,162,963; 5,545,807; 6,300,129;6,255,458; 5,877,397; 5,874,299 and 5,545,806; in PCT publicationsWO91/10741, WO90/04036, and in EP 546073B1 and EP 546073A1.

The transgenic mice described above, referred to herein as “HuMab” mice,contain a human immunoglobulin gene minilocus that encodes unrearrangedhuman heavy (μ and gamma) and kappa light chain immunoglobulinsequences, together with targeted mutations that inactivate theendogenous μ and .kappa. chain loci (Lonberg et al., 1994, Nature 368:856-859).

Accordingly, the aforementioned mice exhibit reduced expression of mouseIgM or kappa and in response to immunization, and the introduced humanheavy and light chain transgenes undergo class switching and somaticmutation to generate high affinity human IgG kappa monoclonal antibodies(Lonberg et al., supra.; Lonberg and Huszar, 1995, Intern. Rev.Immunol., 13: 65-93; Harding and Lonberg, 1995, Ann. N.Y Acad. Sci 764:536-546). The preparation of HuMab mice is described in detail in Tayloret al., 1992, Nucleic Acids Research, 20: 6287-6295; Chen et al., 1993,International Immunology 5: 647-656; Tuaillon et al., 1994, J. Immunol.152: 2912-2920; Lonberg et al., 1994, Nature 368: 856-859; Lonberg,1994, Handbook of Exp. Pharmacology 113: 49-101; Taylor et al., 1994,International Immunology 6: 579-591; Lonberg and Huszar, 1995, Intern.Rev. Immunol. 13: 65-93; Harding and Lonberg, 1995, Ann. N.Y Acad. Sci.764: 536-546; Fishwild et al., 1996, Nature Biotechnology 14: 845-851.See further U.S. Pat. Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425;5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; and 5,770,429; aswell as U.S. Pat. No. 5,545,807; International Publication Nos. WO93/1227; WO 92/22646; and WO 92/03918. Technologies utilized forproducing human antibodies in these transgenic mice are disclosed alsoin WO 98/24893, and Mendez et al., 1997, Nature Genetics 15: 146-156.For example, the HCO7 and HCO12 transgenic mice strains can be used togenerate human anti-DKK1 antibodies.

Using hybridoma technology, antigen-specific human MAbs with the desiredspecificity can be produced and selected from the transgenic mice suchas those described above. Such antibodies may be cloned and expressedusing a suitable vector and host cell, or the antibodies can beharvested from cultured hybridoma cells.

Fully human antibodies can also be derived from phage-display libraries(as disclosed in Hoogenboom et al., 1991, J. Mol. Biol. 227:381; andMarks et al., 1991, J. Mol. Biol. 222:581). Phage display techniquesmimic immune selection through the display of antibody repertoires onthe surface of filamentous bacteriophage, and subsequent selection ofphage by their binding to an antigen of choice. One such technique isdescribed in PCT Publication No. WO99/10494, which describes theisolation of high affinity and functional agonistic antibodies for MPL-and msk-receptors using such an approach.

The anti-DKK1 agents provided herein may also block or reduce bindingbetween DKK1 and LRP5 and/or LRP6, thereby stimulating at least oneactivity associated with Wnt signaling. The agents can be an antibody oran immunologically functional fragment thereof and thus includeantibodies with a naturally occurring structure, as well as polypeptidesthat have an antigen binding domain (e.g., a domain antibody). Theantibodies and fragments can be used to treat a variety of differentdiseases including preventing or treating conditions relating to loss ofbone mass or to stimulate production of new bone, as well as variousnon-bone related disorders. Nucleic acids molecules, vectors, and hostcells useful in the production of the antibodies and selective bindingagents are also provided.

Some of the antibodies and immunologically functional fragments that areprovided include one or more of the following light chain (LC)complementary determining regions (CDRs): (i) a LC CDR1 with at least80% sequence identity to SEQ ID NO: 97, 103, 109, 115, 121, 127, 133,139, 145, 151, 157, 163, 169, 175, 181, 187, 193, 199, 205, 211, 217, or223; (ii) a LC CDR2 with at least 80% sequence identity to SEQ ID NO:98, 104, 110, 116, 122, 128, 134, 139, 146, 152, 158, 164, 170, 176,182, 188, 194, 200, 206, 212, 218, or 224; and (iii) a LC CDR3 with atleast 80% sequence identity to SEQ ID NO: 99, 105, 111, 117, 123, 129,135, 140, 147, 153, 159, 165, 171, 177, 183, 189, 195, 201, 207, 213,219, or 225. Some of the antibodies and immunologically functionalfragments that are provided include one or more of the preceding LC CDRsand/or one or more of the following heavy chain (HC) complementarydetermining regions (CDRs): (i) a HC CDR1 with at least 80% sequenceidentity to SEQ ID NO: 100, 106, 112, 118, 124, 130, 136, 142, 148, 154,160, 166, 172, 178, 184, 190, 196, 202, 208, 214, 220, or 226; (ii) a HCCDR2 with at least 80% sequence identity to SEQ ID NO: 101, 107, 113,119, 125, 131, 137, 143, 149, 155, 161, 167, 173, 179, 185, 191, 197,203, 209, 215, 221, or 227; and (iii) a HC CDR3 with at least 80%sequence identity to SEQ ID NO: 102, 108, 114, 120, 126, 132, 138, 144,150, 156, 162, 168, 174, 180, 186, 192, 198, 204, 210, 216, 222 or 228.Some of the antibodies and immunologically functional fragments thereofthat are provided also include one or more LC CDRs and one or more HCCDRs above.

Such antibodies or fragments can specifically bind a DKK1 polypeptide.Certain antibodies or fragments include one, two, three, four, five orall six of the forgoing CDRs.

The light chain and heavy chains of other antibodies or fragments are asdescribed above but have at least 90% sequence identity to the foregoingsequences. Still other antibodies or fragments thereof are ones having alight chain in which CDR1 has the amino acid sequence as set forth inSEQ ID NOs: 97, 103, 109, 115, 121, 127, 133, 139, 145, 151, 157, 163,169, 175, 181, 187, 193, 199, 205, 211, 217, or 223, CDR2 has the aminoacid sequence as set forth in SEQ ID NOs: 98, 104, 110, 116, 122, 128,134, 139, 146, 152, 158, 164, 170, 176, 182, 188, 194, 200, 206, 212,218, or 224 and/or CDR3 has the amino acid sequence as set forth in SEQID NOs: 99, 105, 111, 117, 123, 129, 135, 140, 147, 153, 159, 165, 171,177, 183, 189, 195, 201, 207, 213, 219, or 225. Some antibodies andfragments may also have a heavy chain in which CDR1 has the amino acidsequence as set forth in SEQ ID NOs: 100, 106, 112, 118, 124, 130, 136,142, 148, 154, 160, 166, 172, 178, 184, 190, 196, 202, 208, 214, 220, or226, CDR2 has the amino acid sequence as set forth in SEQ ID NOs: 101,107, 113, 119, 125, 131, 137, 143, 149, 155, 161, 167, 173, 179, 185,191, 197, 203, 209, 215, 221, or 227 and/or HC CDR3 has the amino acidsequence as set forth in SEQ ID NOs: 102, 108, 114, 120, 126, 132, 138,144, 150, 156, 162, 168, 174, 180, 186, 192, 198, 204, 210, 216, 222 or228. Certain antibodies or fragments include a light chain CDR3 with theamino acid sequence of SEQ ID NOs: 99, 105, 111, 117, 123, 129, 135,140, 147, 153, 159, 165, 171, 177, 183, 189, 195, 201, 207, 213, 219, or225 and/or a heavy chain CDR3 with the amino acid sequence of SEQ IDNOs: 102, 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174,180, 186, 192, 198, 204, 210, 216, 222 or 228.

Certain other antibodies and immunologically functional fragments thatare provided include (a) a light chain variable region (VL) having 80%,85%, 90%, 92%, 95% or greater sequence identity with SEQ ID NOs: 10, 14,18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86,90, or 94; (b) a heavy chain variable region (VH) having at least 80%sequence identity with SEQ ID NOs: 12, 16, 20, 24, 28, 32, 36, 40, 44,48, 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, or 96; or (c) a VL of(a) and a VH of (b).

Other antibodies or fragments are similar in structure but the VL has atleast 90%, 92%, or more preferably 95% sequence identity with SEQ IDNOs: 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74,78, 82, 86, 90, or 94; and the VH has at least 90% sequence identitywith SEQ ID NOs: 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64,68, 72, 76, 80, 84, 88, 92, or 96. In certain antibodies or fragments,the VL has at least 98% sequence identity with SEQ ID NOs: 10, 14, 18,22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90,or 94; and the VH has at least 98% sequence identity with SEQ ID NOs:12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80,84, 88, 92, or 96. Still other antibodies or fragments are ones thatinclude a VL that has the amino acid sequence of SEQ ID NOs: 10, 14, 18,22, 26, 30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90,or 94, and/or a VH that has the amino acid sequence of SEQ ID NOs: 12,16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84,88, 92, or 96.

Some antibodies or fragments include a light chain that comprises orconsists of the amino acid sequence of SEQ ID NOs: 10, 14, 18, 22, 26,30, 34, 38, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86, 90, or 94and/or a heavy chain that comprises or consists of the amino acidsequence of SEQ ID NOs: 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56,60, 64, 68, 72, 76, 80, 84, 88, 92, or 96.

Also included are isolated antibodies or an immunologically functionalfragments thereof that specifically bind a mature human DKK1 proteinexpressed from the sequence depicted in SEQ ID NO: 1, wherein saidantibody binds to an epitope comprising two loops, said loops beingformed by disulfide bonds between amino acids 220 and 237 of SEQ ID NO:2 and between cysteine residues 245 and 263 of SEQ ID NO:2.

Other antibodies or fragments that are disclosed compete with anantibody such as those described above for specific binding to a DKK1polypeptide. For example, some antibodies and fragments compete with anantibody that consists of two identical heavy chains and two identicallight chains, wherein the heavy chains comprise SEQ ID NO: 42 and saidlight chains comprise SEQ ID NO: 44.

The various antibodies and fragments that are provided may include asingle light and/or heavy chain or a single variable light domain and/ora single variable heavy domain. Other antibodies and fragments includetwo light and/or two heavy chains. In those instances in which theantibody or fragment includes two light and/or heavy chains, the twolight chains in some instances are identical to one another; likewise,the two heavy chains in some instances are identical. The antibodiesthat are provided may include, for example, monoclonal antibodies, ahuman antibody, a chimeric antibody, or a humanized antibody. Theimmunologically functional fragments may include, but are not limitedto, a scFv, a Fab, a Fab′, a F(ab′)², or a domain antibody. In certaininstances, the antibody or fragment dissociates from a DKK1 polypeptidewith a k_(d) (k_(off)) of 5×10⁻⁴ or less.

Pharmaceutical compositions that include any of the foregoing antibodiesand immunologically active fragments are also provided. Suchcompositions typically also include a buffer, a pharmaceuticallyacceptable diluent, a carrier, a solubilizer, an emulsifier or apreservative. The use of the foregoing antibodies and immunologicallyactive fragments in the preparation of a pharmaceutical composition ormedicament is also provided.

A variety of nucleic acids encoding the foregoing antibodies are alsoprovided. Some nucleic acids, for instance, encode (a) a light chain CDRwith the amino acid sequence as set forth in SEQ ID NOs: 9, 13, 17, 21,25, 29, 33, 37, 41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89,and/or 93; and/or (b) a heavy chain CDR with the amino acid sequence asset forth in SEQ ID NOs: 11, 15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55,59, 63, 67, 71, 75, 79, 83, 87, 91 and/or 95, such that the encodedCDR(s) encode an antibody or an immunologically functional fragmentthereof that can specifically bind a DKK1 polypeptide. Certain othernucleic acids comprise or consist of a sequence that encodes a variablelight region (VL) and/or a variable heavy region (VH) of an antibody orimmunologically active fragment, wherein the VL has at least 80%, 90% or95% sequence identity with SEQ ID NOs: 9, 13, 17, 21, 25, 29, 33, 37,41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, or 93 and the VH hasat least 80% 90%, or 95% sequence identity with SEQ ID NOs: 11, 15, 19,23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83, 87, 91or 95. Some of the nucleic acids include a sequence that encodes a VLthat comprises or consists of SEQ ID NOs: 9, 13, 17, 21, 25, 29, 33, 37,41, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, or 93 and/or asequence that encodes a VH that comprises or consists of SEQ ID NOs: 11,15, 19, 23, 27, 31, 35, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79, 83,87, 91 or 95. Expression vectors comprising the foregoing nucleic acidsare also disclosed herein, as are cells (e.g., CHO cells) that comprisesuch expression vectors. Methods of producing an antibody or animmunologically active fragment thereof by culturing cells that containsuch expression vectors are also described.

Provided herein are novel DKK1 antibodies that are effective in treatingconditions requiring increased bone building, for example, fracturerepair or bone loss associated with pathological conditions, such asmultiple myeloma. In addition, provided herein are combinations ofagents that increase bone anabolism including combinations of DKK1 andsclerostin inhibitors. These combinations can be used for treatment of,for example, osteoporosis, increase the rate of fracture healing, andany number of conditions requiring an increase in the rate of bonebuilding. The combination therapeutic can take the form of two separateinhibitors, for example, an anti-sclerostin antibody and an anti-DKK1antibody, or can be a single molecular entity, for example, a bispecificantibody.

As used herein, a bispecific antibody binds one antigen on one of itstwo binding arms, and binds a different antigen on its second arm. Thusa bispecific antibody has two distinct antigen binding arms and ismonovalent for each antigen it binds. Bispecific and bifunctional DKK1antibodies provided herein can include one or more CDRs or one or morevariable regions as described above. A bispecific or bifunctionalantibody in some instances is an artificial hybrid antibody having twodifferent heavy/light chain pairs and two different binding sites. Thesebispecific antibodies may be produced by a variety of methods including,but not limited to, fusion of hybridomas or linking of Fab′ fragments.See, e.g., Songsivilai & Lachmann, 1990, Clin. Exp. Immunol. 79:315-321; Kostelny et al., 1992, J. Immunol. 148: 1547-1553.

Bispecific molecules can also be created according to the invention byfusion. In one example, an antibody of the invention can be linked(e.g., by expressing fused proteins, chemical linking, high affinitynon-covalent association or the like) to one or more other bindingmolecules. Examples of such binding molecules include but are notlimited to another antibody, antibody fragment, peptide or bindingmimetic, such that a bispecific molecule results.

Bispecific molecules can also comprise a first binding specificity forsclerostin and a second binding specificity for a second target. Forexample, the second target can be another epitope of sclerostindifferent from the first epitope. Another example is a bispecificmolecule comprising at least one first binding specificity forsclerostin and a second binding specificity for an epitope within DKK1.Another example is a bispecific molecule comprising at least one firstbinding specificity for sclerostin and a second binding specificity foran epitope within LRP4. Additionally, for the invention in which thebispecific molecule is multi-specific, the molecule can further includea third binding specificity, in addition to the first and second targetepitope.

In one embodiment, the bispecific molecules of the invention comprise asa binding specificity at least one antibody, or an antibody fragmentthereof, including, e.g., an Fab, Fab′, F(ab′)₂, Fv, or a single chainFv from a novel anti-DKK1 antibody sequence provided herein. It may alsobe a light chain or heavy chain dimer, or any minimal fragment such as aFv or a single chain construct as described in Ladner eta/. U.S. Pat.No. 4,946,778.

Bispecific molecules can be prepared by chemically conjugating thebinding portions using methods known in the art. When the bindingportions are proteins or peptides, a variety of coupling orcross-linking agents can be used for covalent conjugation. Examplesinclude protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate(SATA), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB),o-phenylenedimaleimide (oPDM),N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), andsulfosuccinimidyl4-(N-maleimidomethyl) cyclohaxane-lcarboxylate(sulfo-SMCC) (see e.g., Karpovsky et a/, 1984 J. Exp. Med. 160:1686;Liu, M A et al., 1985 Proc. Natl. Acad. Sci. USA 82:8648). Other methodsinclude those described in Paulus, 1985 Behring Ins. Mitt. No. 78,118-132; Brennan et al., 1985 Science 229:81-83, and Glennie et al.,1987 J. Immunol. 139: 2367-2375. Conjugating agents include SATA andsulfo-SMCC, both available from Pierce Chemical Co. (Rockford, Ill.).When the binding portions are antibodies, they can be conjugated bysulfhydryl bonding of the hinge regions of the two heavy chains. In oneembodiment, the hinge region is modified to contain an odd number ofsulfhydryl residues such that there is a free sulfhydryl group that hasnot formed a disulfide linkage with a corresponding heavy or light chaincounterpart.

Bispecific molecules may comprise at least two single chain molecules.Non-limiting examples of methods for preparing bispecific molecules aredescribed various patent publications including in U.S. Pat. No.5,260,203; U.S. Pat. No. 5,455,030; U.S. Pat. No. 4,881,175; U.S. Pat.No. 5,132,405; U.S. Pat. No. 5,091,513; U.S. Pat. No. 5,476,786; U.S.Pat. No. 5,013,653; U.S. Pat. No. 5,258,498; U.S. Pat. No. 5,482,858;and U.S. Patent Application No. 2010/0076178.

Examples of partners for either combination therapy with DKK1 inhibitorsor bi- or multi-specific molecules including DKK1 binding portionsinclude sclerostin antibodies or binding fragments that specificallyrecognize sclerostin proteins. Sclerostin has been previously describedas being involved in regulating bone density through wnt signalingpathways (PCT WO 06/119107).

There is a report of a combination of a DKK1 antibody and a sclerostinantibody where it is suggested that this combination can increase thebone mineral density of cancellous or spongy bone more than either alonein model animals (PCT WO 09/047356) and improved the increase in totalbone mineral content, density and cortical thickness. However, in thoseexamples intact bone was used, not fractured bone.

Reports indicate that DKK1 expression is elevated in fracture models ofnon-unions (Bajada, et al., 2009 Bone; 45(4):726-35.). Likewise, healthybone expresses lower levels of DKK1 helping to explain the limitedeffect of DKK1 antibodies alone on BMD in intact bone (see Example 15).Thus, combinations of sclerostin and DKK1 inhibitors to treat fracturesare particularly useful given the surprisingly strong healing responseincluding the significant increase in the peak load in a relativelyshort period.

Variants

Some of the antibodies or immunologically functional fragments that areprovided are variant forms of the antibodies and fragments disclosedabove (e.g., those having the sequences listed in Table 1). Forinstance, some of the antibodies or fragments are ones having one ormore conservative amino acid substitutions in one or more of the heavyor light chains, variable regions or CDRs listed in Table 1.

Naturally-occurring amino acids may be divided into classes based oncommon side chain properties: [0149] 1) hydrophobic: norleucine, Met,Ala, Val, Leu, Ile; [0150] 2) neutral hydrophilic: Cys, Ser, Thr, Asn,Gln; [0151] 3) acidic: Asp, Glu; [0152] 4) basic: His, Lys, Arg; [0153]5) residues that influence chain orientation: Gly, Pro; and [0154] 6)aromatic: Trp, Tyr, Phe. Conservative amino acid substitutions mayinvolve exchange of a member of one of these classes with another memberof the same class. Conservative amino acid substitutions may encompassnon-naturally occurring amino acid residues, which are typicallyincorporated by chemical peptide synthesis rather than by synthesis inbiological systems. These include peptidomimetics and other reversed orinverted forms of amino acid moieties.

Non-conservative substitutions may involve the exchange of a member ofone of the above classes for a member from another class. Suchsubstituted residues may be introduced into regions of the antibody thatare homologous with human antibodies, or into the non-homologous regionsof the molecule.

In making such changes, according to certain embodiments, thehydropathic index of amino acids may be considered. The hydropathicprofile of a protein is calculated by assigning each amino acid anumerical value (“hydropathy index”) and then repetitively averagingthese values along the peptide chain. Each amino acid has been assigneda hydropathic index on the basis of its hydrophobicity and chargecharacteristics. They are: isoleucine (+4.5); valine (+4.2); leucine(+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine(+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

The importance of the hydropathic profile in conferring interactivebiological function on a protein is understood in the art (see, forexample, Kyte et al., 1982, J. Mol. Biol. 157:105-131). It is known thatcertain amino acids may be substituted for other amino acids having asimilar hydropathic index or score and still retain a similar biologicalactivity. In making changes based upon the hydropathic index, in certainembodiments, the substitution of amino acids whose hydropathic indicesare within .+−0.2 is included. In some aspects of the invention, thosewhich are within .+−0.1 are included, and in other aspects of theinvention, those within .+−0.0.5 are included.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity,particularly where the biologically functional protein or peptidethereby created is intended for use in immunological embodiments, as inthe present case. In certain embodiments, the greatest local averagehydrophilicity of a protein, as governed by the hydrophilicity of itsadjacent amino acids, correlates with its immunogenicity andantigen-binding or immunogenicity, that is, with a biological propertyof the protein.

The following hydrophilicity values have been assigned to these aminoacid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0.+−0.1);glutamate (+3.0.+−0.1); serine (+0.3); asparagine (+0.2); glutamine(+0.2); glycine (0); threonine (−0.4); proline (−0.5.+−0.1); alanine(−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine(−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3);phenylalanine (−2.5) and tryptophan (−3.4). In making changes based uponsimilar hydrophilicity values, in certain embodiments, the substitutionof amino acids whose hydrophilicity values are within .+/−.2 isincluded, in other embodiments, those which are within .+/−.1 areincluded, and in still other embodiments, those within .+/−.0.5 areincluded. In some instances, one may also identify epitopes from primaryamino acid sequences on the basis of hydrophilicity. These regions arealso referred to as “epitopic core regions.”

A skilled artisan will be able to determine suitable variants ofpolypeptides as set forth herein using well-known techniques. Oneskilled in the art may identify suitable areas of the molecule that maybe changed without destroying activity by targeting regions not believedto be important for activity. The skilled artisan also will be able toidentify residues and portions of the molecules that are conserved amongsimilar polypeptides. In further embodiments, even areas that may beimportant for biological activity or for structure may be subject toconservative amino acid substitutions without destroying the biologicalactivity or without adversely affecting the polypeptide structure.

Additionally, one skilled in the art can review structure-functionstudies identifying residues in similar polypeptides that are importantfor activity or structure. In view of such a comparison, one can predictthe importance of amino acid residues in a protein that correspond toamino acid residues important for activity or structure in similarproteins. One skilled in the art may opt for chemically similar aminoacid substitutions for such predicted important amino acid residues.

One skilled in the art can also analyze the three-dimensional structureand amino acid sequence in relation to that structure in similarpolypeptides. In view of such information, one skilled in the art maypredict the alignment of amino acid residues of an antibody with respectto its three dimensional structure. One skilled in the art may choosenot to make radical changes to amino acid residues predicted to be onthe surface of the protein, since such residues may be involved inimportant interactions with other molecules. Moreover, one skilled inthe art may generate test variants containing a single amino acidsubstitution at each desired amino acid residue. These variants can thenbe screened using assays for DKK1 neutralizing activity, (see examplesbelow) thus yielding information regarding which amino acids can bechanged and which must not be changed. In other words, based oninformation gathered from such routine experiments, one skilled in theart can readily determine the amino acid positions where furthersubstitutions should be avoided either alone or in combination withother mutations.

A number of scientific publications have been devoted to the predictionof secondary structure. See Moult, 1996, Curr. Op. in Biotech.7:422-427; Chou et al., 1974, Biochemistry 13:222-245; Chou et al.,1974, Biochemistry 113:211-222; Chou et al., 1978, Adv. Enzymol. Relat.Areas Mol. Biol. 47:45-148; Chou et al., 1979, Ann. Rev. Biochem.47:251-276; and Chou et al., 1979, Biophys. J. 26:367-384. Moreover,computer programs are currently available to assist with predictingsecondary structure. One method of predicting secondary structure isbased upon homology modeling. For example, two polypeptides or proteinsthat have a sequence identity of greater than 30%, or similarity greaterthan 40% often have similar structural topologies. The recent growth ofthe protein structural database (PDB) has provided enhancedpredictability of secondary structure, including the potential number offolds within a polypeptide's or protein's structure. See Holm et al.,1999, Nucl. Acid. Res. 27:244-247. It has been suggested (Brenner etal., 1997, Curr. Op. Struct. Biol. 7:369-376) that there are a limitednumber of folds in a given polypeptide or protein and that once acritical number of structures have been resolved, structural predictionwill become dramatically more accurate.

Additional methods of predicting secondary structure include “threading”(Jones, 1997, Curr. Opin. Struct. Biol. 7:377-87; Sippl et al., 1996,Structure 4:15-19), “profile analysis” (Bowie et al., 1991, Science253:164-170; Gribskov et al., 1990, Meth. Enzym. 183:146-159; Gribskovet al., 1987, Proc. Nat. Acad. Sci. 84:4355-4358), and “evolutionarylinkage” (See Holm, 1999, supra; and Brenner, 1997, supra).

In some embodiments of the invention, amino acid substitutions are madethat: (1) reduce susceptibility to proteolysis, (2) reducesusceptibility to oxidation, (3) alter binding affinity for formingprotein complexes, (4) alter ligand or antigen binding affinities,and/or (4) confer or modify other physicochemical or functionalproperties on such polypeptides. For example, single or multiple aminoacid substitutions (in certain embodiments, conservative amino acidsubstitutions) may be made in the naturally-occurring sequence.Substitutions can be made in that portion of the antibody that liesoutside the domain(s) forming intermolecular contacts). In suchembodiments, conservative amino acid substitutions can be used that donot substantially change the structural characteristics of the parentsequence (e.g., one or more replacement amino acids that do not disruptthe secondary structure that characterizes the parent or nativeantibody). Examples of art-recognized polypeptide secondary and tertiarystructures are described in Proteins, Structures and MolecularPrinciples (Creighton, Ed.), 1984, W. H. New York: Freeman and Company;Introduction to Protein Structure (Branden and Tooze, eds.), 1991, NewYork: Garland Publishing; and Thornton et at., 1991, Nature 354: 105,which are each incorporated herein by reference.

The invention also encompasses glycosylation variants of the inventiveantibodies wherein the number and/or type of glycosylation site(s) hasbeen altered compared to the amino acid sequences of the parentpolypeptide. In certain embodiments, antibody protein variants comprisea greater or a lesser number of N-linked glycosylation sites than thenative antibody. An N-linked glycosylation site is characterized by thesequence: Asn-X-Ser or Asn-X-Thr, wherein the amino acid residuedesignated as X may be any amino acid residue except proline. Thesubstitution of amino acid residues to create this sequence provides apotential new site for the addition of an N-linked carbohydrate chain.Alternatively, substitutions that eliminate or alter this sequence willprevent addition of an N-linked carbohydrate chain present in the nativepolypeptide. For example, the glycosylation can be reduced by thedeletion of an Asn or by substituting the Asn with a different aminoacid. In other embodiments, one or more new N-linked sites are created.Antibodies typically have a N-linked glycosylation site in the Fcregion.

Additional preferred antibody variants include cysteine variants whereinone or more cysteine residues in the parent or native amino acidsequence are deleted from or substituted with another amino acid (e.g.,serine). Cysteine variants are useful, inter alia when antibodies mustbe refolded into a biologically active conformation. Cysteine variantsmay have fewer cysteine residues than the native antibody, and typicallyhave an even number to minimize interactions resulting from unpairedcysteines.

The heavy and light chains, variable regions domains and CDRs that aredisclosed can be used to prepare polypeptides that contain an antigenbinding region that can specifically bind to a DKK1 polypeptide. Forexample, one or more of the CDRs listed in Table 1 can be incorporatedinto a molecule (e.g., a polypeptide) covalently or noncovalently tomake an immunoadhesion. An immunoadhesion may incorporate the CDR(s) aspart of a larger polypeptide chain, may covalently link the CDR(s) toanother polypeptide chain, or may incorporate the CDR(s) noncovalently.The CDR(s) enable the immunoadhesion to bind specifically to aparticular antigen of interest (e.g., a DKK1 polypeptide or epitopethereof).

Mimetics (e.g., peptide mimetics” or “peptidomimetics”) based upon thevariable region domains and CDRs that are described herein are alsoprovided. These analogs can be peptides, non-peptides or combinations ofpeptide and non-peptide regions. Fauchere, 1986, Adv. Drug Res. 15: 29;Veber and Freidinger, 1985, TINS p. 392; and Evans et al., 1987, J. Med.Chem. 30: 1229, which are incorporated herein by reference for anypurpose. Peptide mimetics that are structurally similar totherapeutically useful peptides may be used to produce a similartherapeutic or prophylactic effect. Such compounds are often developedwith the aid of computerized molecular modeling. Generally,peptidomimetics of the invention are proteins that are structurallysimilar to an antibody displaying a desired biological activity, such ashere the ability to specifically bind DKK1, but have one or more peptidelinkages optionally replaced by a linkage selected from: —CH2NH—,—CH2S—, —CH2-CH.2-, —CH—CH— (cis and trans), —COCH2-, —CH(OH)CH2-, and—CH2SO—, by methods well known in the art. Systematic substitution ofone or more amino acids of a consensus sequence with a D-amino acid ofthe same type (e.g., D-lysine in place of L-lysine) may be used incertain embodiments of the invention to generate more stable proteins.In addition, constrained peptides comprising a consensus sequence or asubstantially identical consensus sequence variation may be generated bymethods known in the art (Rizo and Gierasch, 1992, Ann. Rev. Biochem.61: 387), incorporated herein by reference), for example, by addinginternal cysteine residues capable of forming intramolecular disulfidebridges which cyclize the peptide.

Derivatives of the antibodies and immunologically functional fragmentsthat are described herein are also provided. The derivatized antibody orfragment may comprise any molecule or substance that imparts a desiredproperty to the antibody or fragment, such as increased half-life in aparticular use. The derivatized antibody can comprise, for example, adetectable (or labeling) moiety (e.g., a radioactive, colorimetric,antigenic or enzymatic molecule, a detectable bead (such as a magneticor electrodense (e.g., gold) bead), or a molecule that binds to anothermolecule (e.g., biotin or streptavidin)), a therapeutic or diagnosticmoiety (e.g., a radioactive, cytotoxic, or pharmaceutically activemoiety), or a molecule that increases the suitability of the antibodyfor a particular use (e.g., administration to a subject, such as a humansubject, or other in vivo or in vitro uses). Examples of molecules thatcan be used to derivatize an antibody include albumin (e.g., human serumalbumin) and polyethylene glycol (PEG). Albumin-linked and PEGylatedderivatives of antibodies can be prepared using techniques well known inthe art. In one embodiment, the antibody is conjugated or otherwiselinked to transthyretin (TTR) or a TTR variant. The TTR or TTR variantcan be chemically modified with, for example, a chemical selected fromthe group consisting of dextran, poly(n-vinyl pyrrolidone), polyethyleneglycols, propropylene glycol homopolymers, polypropylene oxide/ethyleneoxide co-polymers, polyoxyethylated polyols and polyvinyl alcohols.

Other derivatives include covalent or aggregative conjugates ofanti-DKK1 antibodies, or fragments thereof, with other proteins orpolypeptides, such as by expression of recombinant fusion proteinscomprising heterologous polypeptides fused to the N-terminus orC-terminus of an anti-DKK1 antibody polypeptide. For example, theconjugated peptide may be a heterologous signal (or leader) polypeptide,e.g., the yeast alpha-factor leader, or a peptide such as an epitopetag. Anti-DKK1 antibody-containing fusion proteins can comprise peptidesadded to facilitate purification or identification of the anti-DKK1antibody (e.g., poly-His). An anti-DKK1 antibody polypeptide also can belinked to the FLAG peptide as described in Hopp et al., Bio/Technology6:1204, 1988, and U.S. Pat. No. 5,011,912. The FLAG peptide is highlyantigenic and provides an epitope reversibly bound by a specificmonoclonal antibody (mAb), enabling rapid assay and facile purificationof expressed recombinant protein. Reagents useful for preparing fusionproteins in which the FLAG peptide is fused to a given polypeptide arecommercially available (Sigma, St. Louis, Mo.).

Oligomers that contain one or more anti-DKK1 antibody polypeptides maybe employed as DKK1 antagonists. Oligomers may be in the form ofcovalently-linked or non-covalently-linked dimers, trimers, or higher.Oligomers comprising two or more anti-DKK1 antibody polypeptides arecontemplated for use, with one example being a homodimer. Otheroligomers include heterodimers, homotrimers, heterotrimers,homotetramers, heterotetramers, etc.

One embodiment is directed to oligomers comprising multiple anti-DKK1antibody polypeptides joined via covalent or non-covalent interactionsbetween peptide moieties fused to the anti-DKK1 antibody polypeptides.Such peptides may be peptide linkers (spacers), or peptides that havethe property of promoting oligomerization. Leucine zippers and certainpolypeptides derived from antibodies are among the peptides that canpromote oligomerization of anti-DKK1 antibody polypeptides attachedthereto, as described in more detail below.

In particular embodiments, the oligomers comprise from two to fouranti-DKK1 antibody polypeptides. The anti-DKK1 antibody moieties of theoligomer may be in any of the forms described above, e.g., variants orfragments. Preferably, the oligomers comprise anti-DKK1 antibodypolypeptides that have DKK1 binding activity.

In one embodiment, an oligomer is prepared using polypeptides derivedfrom immunoglobulins. Preparation of fusion proteins comprising certainheterologous polypeptides fused to various portions of antibody-derivedpolypeptides (including the Fc domain) has been described, e.g., byAshkenazi et al., 1991, PNAS USA 88:10535; Byrn et al., 1990, Nature344:677; and Hollenbaugh et al., 1992 “Construction of ImmunoglobulinFusion Proteins”, in Current Protocols in Immunology, Suppl. 4, pages10.19.1-10.19.11.

One embodiment of the present invention is directed to a dimercomprising two fusion proteins created by fusing a DKK1 binding fragmentof an anti-DKK1 antibody to the Fc region of an antibody. The dimer canbe made by, for example, inserting a gene fusion encoding the fusionprotein into an appropriate expression vector, expressing the genefusion in host cells transformed with the recombinant expression vector,and allowing the expressed fusion protein to assemble much like antibodymolecules, whereupon interchain disulfide bonds form between the Fcmoieties to yield the dimer.

The term “Fc polypeptide” as used herein includes native and muteinforms of polypeptides derived from the Fc region of an antibody.Truncated forms of such polypeptides containing the hinge region thatpromotes dimerization also are included. Fusion proteins comprising Fcmoieties (and oligomers formed therefrom) offer the advantage of facilepurification by affinity chromatography over Protein A or Protein Gcolumns.

One suitable Fc polypeptide, described in PCT application WO 93/10151and U.S. Pat. Nos. 5,426,048 and 5,262,522 (each of which is herebyincorporated by reference), is a single chain polypeptide extending fromthe N-terminal hinge region to the native C-terminus of the Fc region ofa human IgG1 antibody. Another useful Fc polypeptide is the Fc muteindescribed in U.S. Pat. No. 5,457,035 and in Baum et al., 1994, EMBO J.13:3992-4001. The amino acid sequence of this mutein is identical tothat of the native Fc sequence presented in WO 93/10151, except thatamino acid 19 has been changed from Leu to Ala, amino acid 20 has beenchanged from Leu to Glu, and amino acid 22 has been changed from Gly toAla. The mutein exhibits reduced affinity for Fc receptors.

In other embodiments, the variable portion of the heavy and/or lightchains of an anti-DKK1 antibody such as disclosed herein may besubstituted for the variable portion of an antibody heavy and/or lightchain.

Alternatively, the oligomer is a fusion protein comprising multipleanti-DKK1 antibody polypeptides, with or without peptide linkers (spacerpeptides). Among the suitable peptide linkers are those described inU.S. Pat. Nos. 4,751,180 and 4,935,233.

Another method for preparing oligomeric anti-DKK1 antibody derivativesinvolves use of a leucine zipper. Leucine zipper domains are peptidesthat promote oligomerization of the proteins in which they are found.Leucine zippers were originally identified in several DNA-bindingproteins (Landschulz et al., 1988, Science 240:1759), and have sincebeen found in a variety of different proteins. Among the known leucinezippers are naturally occurring peptides and derivatives thereof thatdimerize or trimerize. Examples of leucine zipper domains suitable forproducing soluble oligomeric proteins are described in PCT applicationWO 94/10308, and the leucine zipper derived from lung surfactant proteinD (SPD) described in Hoppe et al., 1994, FEBS Letters 344:191, herebyincorporated by reference. The use of a modified leucine zipper thatallows for stable trimerization of a heterologous protein fused theretois described in Fanslow et al., 1994, Semin. Immunol. 6:267-78. In oneapproach, recombinant fusion proteins comprising an anti-DKK1 antibodyfragment or derivative fused to a leucine zipper peptide are expressedin suitable host cells, and the soluble oligomeric anti-DKK1 antibodyfragments or derivatives that form are recovered from the culturesupernatant.

Some antibodies that are provided have a binding affinity (K_(a)) forDKK1 of at least 10⁴ or 10⁵/M x seconds measured, for instance, asdescribed in the examples below. Other antibodies have a k_(a) of atleast 10⁶, 10⁷, 10⁸ or 10⁹/M x seconds. Certain antibodies that areprovided have a low disassociation rate. Some antibodies, for instance,have a K_(off) of 1×10⁻⁴s⁻¹, 1×10⁻⁵s⁻¹ or lower.

In another aspect, the present invention provides an anti-DKK1 antibodyhaving a half-life of at least one day in vitro or in vivo (e.g., whenadministered to a human subject). In one embodiment, the antibody has ahalf-life of at least three days. In another embodiment, the antibody orportion thereof has a half-life of four days or longer. In anotherembodiment, the antibody or portion thereof has a half-life of eightdays or longer. In another embodiment, the antibody or antigen-bindingportion thereof is derivatized or modified such that it has a longerhalf-life as compared to the underivatized or unmodified antibody. Inanother embodiment, the antibody contains point mutations to increaseserum half life, such as described in WO 00/09560.

Nucleic acids that encode one or both chains of an antibody of theinvention, or a fragment, derivative, mutein, or variant thereof,polynucleotides sufficient for use as hybridization probes, PCR primersor sequencing primers for identifying, analyzing, mutating or amplifyinga polynucleotide encoding a polypeptide, anti-sense nucleic acids forinhibiting expression of a polynucleotide, and complementary sequencesof the foregoing are also provided. The nucleic acids can be any length.They can be, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75,100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1,000,1,500, 3,000, 5,000 or more nucleotides in length, and/or can compriseone or more additional sequences, for example, regulatory sequences,and/or be part of a larger nucleic acid, for example, a vector. Thenucleic acids can be single-stranded or double-stranded and can compriseRNA and/or DNA nucleotides, and artificial variants thereof (e.g.,peptide nucleic acids).

Nucleic acids that encode the epitope to which certain of the antibodiesprovided herein bind are also provided. Thus, some nucleic acids encodeamino acids 221-229 and/or 246-253 of SEQ ID NO:2 are included, as arenucleic acids that encode amino acids 221-236 and/or 246-262 of SEQ IDNO:2 and those that encode amino acids 221 to 262 of SEQ ID NO:2 oramino acids 221-253 of SEQ ID NO:2. Nucleic acids encoding fusionproteins that include these peptides are also provided.

DNA encoding antibody polypeptides (e.g., heavy or light chain, variabledomain only, or full length) may be isolated from B-cells of mice thathave been immunized with DKK1 or an immunogenic fragment thereof. TheDNA may be isolated by conventional procedures such as polymerase chainreaction (PCR). Phage display is another example of a known techniquewhereby derivatives of antibodies may be prepared. In one approach,polypeptides that are components of an antibody of interest areexpressed in any suitable recombinant expression system, and theexpressed polypeptides are allowed to assemble to form antibodymolecules.

Exemplary nucleic acids that encode the light and heavy chains, variableregions and CDRs of the antibodies and immunologically functionalfragments that are provided are listed in Table 1 above. Due to thedegeneracy of the genetic code, each of the polypeptide sequences listedin Table 1 is also encoded by a large number of other nucleic acidsequences besides those listed in Table 1. The present inventionprovides each degenerate nucleotide sequence encoding each antibody ofthe invention.

The invention further provides nucleic acids that hybridize to othernucleic acids (e.g., nucleic acids comprising a nucleotide sequencelisted in Tables 1-3) under particular hybridization conditions. Methodsfor hybridizing nucleic acids are well-known in the art. See, e.g.,Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989),6.3.1-6.3.6. As defined herein, a moderately stringent hybridizationcondition uses a prewashing solution containing 5.times. sodiumchloride/sodium citrate (SSC), 0.5% SDS, 1.0 mM EDTA (pH 8.0),hybridization buffer of about 50% formamide, 6.times.SSC, and ahybridization temperature of 55° C. (or other similar hybridizationsolutions, such as one containing about 50% formamide, with ahybridization temperature of 42.degree. C.), and washing conditions of60° C., in 0.5×SSC, 0.1% SDS. A stringent hybridization conditionhybridizes in 6×SSC at 45° C., followed by one or more washes in0.1×SSC, 0.2% SDS at 68° C. Furthermore, one of skill in the art canmanipulate the hybridization and/or washing conditions to increase ordecrease the stringency of hybridization such that nucleic acidscomprising nucleotide sequences that are at least 65, 70, 75, 80, 85,90, 95, 98 or 99% identical to each other typically remain hybridized toeach other.

The basic parameters affecting the choice of hybridization conditionsand guidance for devising suitable conditions are set forth by, forexample, Sambrook, Fritsch, and Maniatis (1989, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., chapters 9 and 11; and Current Protocols in MolecularBiology, 1995, Ausubel et al., eds., John Wiley & Sons, Inc., sections2.10 and 6.3-6.4), and can be readily determined by those havingordinary skill in the art based on, for example, the length and/or basecomposition of the DNA.

Changes can be introduced by mutation into a nucleic acid, therebyleading to changes in the amino acid sequence of a polypeptide (e.g., anantibody or antibody derivative of the invention) that it encodes.Mutations can be introduced using any technique known in the art. In oneembodiment, one or more particular amino acid residues are changedusing, for example, a site-directed mutagenesis protocol. In anotherembodiment, one or more randomly selected residue is changed using, forexample, a random mutagenesis protocol. However it is made, a mutantpolypeptide can be expressed and screened for a desired property.

Mutations can be introduced into a nucleic acid without significantlyaltering the biological activity of a polypeptide that it encodes. Forexample, one can make nucleotide substitutions leading to amino acidsubstitutions at non-essential amino acid residues. Alternatively, oneor more mutations can be introduced into a nucleic acid that selectivelychange the biological activity of a polypeptide that it encodes. Forexample, the mutation can quantitatively or qualitatively change thebiological activity. Examples of quantitative changes includeincreasing, reducing or eliminating the activity. Examples ofqualitative changes include changing the antigen specificity of anantibody.

In another aspect, the present invention provides nucleic acid moleculesthat are suitable for use as primers or hybridization probes for thedetection of nucleic acid sequences of the invention. A nucleic acidmolecule of the invention can comprise only a portion of a nucleic acidsequence encoding a full-length polypeptide of the invention, forexample, a fragment that can be used as a probe or primer or a fragmentencoding an active portion (e.g., a DKK1 binding portion) of apolypeptide of the invention.

Probes based on the sequence of a nucleic acid of the invention can beused to detect the nucleic acid or similar nucleic acids, for example,transcripts encoding a polypeptide of the invention. The probe cancomprise a label group, e.g., a radioisotope, a fluorescent compound, anenzyme, or an enzyme co-factor. Such probes can be used to identify acell that expresses

In another aspect, the present invention provides vectors comprising anucleic acid encoding a polypeptide of the invention or a portionthereof (e.g., a fragment containing one or more CDRs or one or morevariable region domains). Examples of vectors include, but are notlimited to, plasmids, viral vectors, non-episomal mammalian vectors andexpression vectors, for example, recombinant expression vectors. Therecombinant expression vectors of the invention can comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell. The recombinant expression vectors include one ormore regulatory sequences, selected on the basis of the host cells to beused for expression, which is operably linked to the nucleic acidsequence to be expressed. Regulatory sequences include those that directconstitutive expression of a nucleotide sequence in many types of hostcells (e.g., SV40 early gene enhancer, Rous sarcoma virus promoter andcytomegalovirus promoter), those that direct expression of thenucleotide sequence only in certain host cells (e.g., tissue-specificregulatory sequences, see Voss et al., 1986, Trends Biochem. Sci.11:287, Maniatis et al., 1987, Science 236:1237, incorporated byreference herein in their entireties), and those that direct inducibleexpression of a nucleotide sequence in response to particular treatmentor condition (e.g., the metallothionin promoter in mammalian cells andthe tet-responsive and/or streptomycin responsive promoter in bothprokaryotic and eukaryotic systems (see id.). It will be appreciated bythose skilled in the art that the design of the expression vector candepend on such factors as the choice of the host cell to be transformed,the level of expression of protein desired, etc. The expression vectorsof the invention can be introduced into host cells to thereby produceproteins or peptides, including fusion proteins or peptides, encoded bynucleic acids as described herein.

In another aspect, the present invention provides host cells into whicha recombinant expression vector of the invention has been introduced. Ahost cell can be any prokaryotic cell (for example, E. coli) oreukaryotic cell (for example, yeast, insect, or mammalian cells (e.g.,CHO cells)). Vector DNA can be introduced into prokaryotic or eukaryoticcells via conventional transformation or transfection techniques. Forstable transfection of mammalian cells, it is known that, depending uponthe expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., for resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin and methotrexate. Cells stablytransfected with the introduced nucleic acid can be identified by drugselection (e.g., cells that have incorporated the selectable marker genewill survive, while the other cells die), among other methods.

The non-human antibodies that are provided can be, for example, derivedfrom any antibody-producing animal, such as mouse, rat, rabbit, goat,donkey, or non-human primate (such as monkey (e.g., cynomologous orrhesus monkey) or ape (e.g., chimpanzee)). Non-human antibodies can beused, for instance, in in vitro cell culture and cell-culture basedapplications, or any other application where an immune response to theantibody does not occur or is insignificant, can be prevented, is not aconcern, or is desired. In certain embodiments of the invention, theantibodies may be produced by immunizing with full-length DKK1 or withthe carboxy-terminal half of DKK1. Alternatively, the certain non-humanantibodies may be raised by immunizing with amino acids 221-236 and/oramino acids 246-262 of SEQ ID NO:2, which are segments of human DKK1that form part of the epitope to which certain antibodies providedherein bind (e.g., the 11H10, see FIG. 1). The antibodies may bepolyclonal, monoclonal, or may be synthesized in host cells byexpressing recombinant DNA.

Fully human antibodies may be prepared as described above by immunizingtransgenic animals containing human immunoglobulin loci or by selectinga phage display library that is expressing a repertoire of humanantibodies.

The monoclonal antibodies (mAbs) of the invention can be produced by avariety of techniques, including conventional monoclonal antibodymethodology, e.g., the standard somatic cell hybridization technique ofKohler and Milstein, 1975, Nature 256: 495. Alternatively, othertechniques for producing monoclonal antibodies can be employed, forexample, the viral or oncogenic transformation of B-lymphocytes. Onesuitable animal system for preparing hybridomas is the murine system,which is a very well established procedure. Immunization protocols andtechniques for isolation of immunized splenocytes for fusion are knownin the art. For such procedures, B cells from immunized mice are fusedwith a suitable immortalized fusion partner, such as a murine myelomacell line. If desired, rats or other mammals besides can be immunizedinstead of mice and B cells from such animals can be fused with themurine myeloma cell line to form hybridomas. Alternatively, a myelomacell line from a source other than mouse may be used. Fusion proceduresfor making hybridomas also are well known.

The single chain antibodies that are provided may be formed by linkingheavy and light chain variable domain (Fv region) fragments (see, e.g.,Table 1) via an amino acid bridge (short peptide linker), resulting in asingle polypeptide chain. Such single-chain Fvs (scFvs) may be preparedby fusing DNA encoding a peptide linker between DNAs encoding the twovariable domain polypeptides (VL and VH). The resulting polypeptides canfold back on themselves to form antigen-binding monomers, or they canform multimers (e.g., dimers, trimers, or tetramers), depending on thelength of a flexible linker between the two variable domains (Kortt etal., 1997, Prot. Eng. 10:423; Kortt et al., 2001, Biomol. Eng.18:95-108). By combining different VL and VH-comprising polypeptides,one can form multimeric scFvs that bind to different epitopes (Kriangkumet al., 2001, Biomol. Eng. 18:31-40). Techniques developed for theproduction of single chain antibodies include those described in U.S.Pat. No. 4,946,778; Bird, 1988, Science 242:423; Huston et al., 1988,Proc. Natl. Acad. Sci. USA 85:5879; Ward et al., 1989, Nature 334:544,de Graaf et al., 2002, Methods Mol Biol. 178:379-87.

Antibodies provided herein that are of one subclass can be changed toantibodies from a different subclass using subclass switching methods.For example, the variable domains depicted in Table 1 can be attached toconstant domains of any desired Ig subtype. Such techniques allow thepreparation of new antibodies that possess the antigen-bindingproperties of a given antibody (the parent antibody), but also exhibitbiological properties associated with an antibody isotype or subclassdifferent from that of the parent antibody. Recombinant DNA techniquesmay be employed. Cloned DNA encoding particular antibody polypeptidesmay be employed in such procedures, e.g., DNA encoding the constantdomain of an antibody of the desired isotype. See, e.g., Lantto et al.,2002, Methods Mol. Biol. 178:303-16.

Accordingly, the antibodies that are provided include a desired isotype(for example, IgA, IgG1, IgG2, IgG3, IgG4, IgE, and IgD) as well as Fabor F(ab′) 2 fragments thereof. Moreover, if an IgG4 is desired, it mayalso be desired to introduce a point mutation in the hinge region asdescribed in Bloom et al., 1997, Protein Science 6:407) to alleviate atendency to form intra-H chain disulfide bonds that can lead toheterogeneity in the IgG4 antibodies.

Moreover, techniques for deriving antibodies having different properties(i.e., varying affinities for the antigen to which they bind) are alsoknown. One such technique, referred to as chain shuffling, involvesdisplaying immunoglobulin variable domain gene repertoires on thesurface of filamentous bacteriophage, often referred to as phagedisplay. Chain shuffling has been used to prepare high affinityantibodies to the hapten 2-phenyloxazol-5-one, as described by Marks etal., 1992, BioTechnology, 10:779.

Conservative modifications may be made to the heavy and light chainsdescribed in Table 1 (and corresponding modifications to the encodingnucleic acids) to produce an anti-DKK1 antibody having functional andbiochemical characteristics. Methods for achieving such modificationsare described above.

Antibodies and functional fragments thereof according to the inventionmay be further modified in various ways. For example, if they are to beused for therapeutic purposes, they may be conjugated with polyethyleneglycol (pegylated) to prolong the serum half-life or to enhance proteindelivery. Alternatively, the V region of the subject antibodies orfragments thereof may be fused with the Fc region of a differentantibody molecule. The Fc region used for this purpose may be modifiedso that it does not bind complement, thus reducing the likelihood ofinducing cell lysis in the patient when the fusion protein is used as atherapeutic agent. In addition, the subject antibodies or functionalfragments thereof may be conjugated with human serum albumin to enhancethe serum half-life of the antibody or fragment thereof. Another usefulfusion partner for the inventive antibodies or fragments thereof istransthyretin (TTR). TTR has the capacity to form a tetramer, thus anantibody-TTR fusion protein can form a multivalent antibody which mayincrease its binding avidity.

Alternatively, substantial modifications in the functional and/orbiochemical characteristics of the antibodies and fragments describedherein may be achieved by creating substitutions in the amino acidsequence of the heavy and light chains that differ significantly intheir effect on maintaining (a) the structure of the molecular backbonein the area of the substitution, for example, as a sheet or helicalconformation, (b) the charge or hydrophobicity of the molecule at thetarget site, or (c) the bulkiness of the side chain. A “conservativeamino acid substitution” may involve a substitution of a native aminoacid residue with a nonnative residue that has little or no effect onthe polarity or charge of the amino acid residue at that position.Furthermore, any native residue in the polypeptide may also besubstituted with alanine, as has been previously described for alaninescanning mutagenesis.

Amino acid substitutions (whether conservative or non-conservative) ofthe subject antibodies can be implemented by those skilled in the art byapplying routine techniques. Amino acid substitutions can be used toidentify important residues of the antibodies provided herein, or toincrease or decrease the affinity of these antibodies for human DKK1 orfor modifying the binding affinity of other anti-DKK1 antibodiesdescribed herein.

The anti-DKK1 antibodies and immunological functional fragments can beprepared by any of a number of conventional techniques. For example,anti-DKK1 antibodies may be produced by recombinant expression systems,using any technique known in the art. See, for example, MonoclonalAntibodies, Hybridomas: A New Dimension in Biological Analyses, Kennetet al. (eds.) Plenum Press, New York (1980): and Antibodies: ALaboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1988).

Antibodies of the present invention can be expressed in hybridoma celllines or in cell lines other than hybridomas. Expression constructsencoding the antibodies can be used to transform a mammalian, insect ormicrobial host cell. Transformation can be performed using any knownmethod for introducing polynucleotides into a host cell, including, forexample packaging the polynucleotide in a virus or bacteriophage andtransducing a host cell with the construct by transfection proceduresknown in the art, as exemplified by U.S. Pat. Nos. 4,399,216, 4,912,040,4,740,461, and 4,959,455. The optimal transformation procedure used willdepend upon which type of host cell is being transformed. Methods forintroduction of heterologous polynucleotides into mammalian cells arewell known in the art and include, but are not limited to,dextran-mediated transfection, calcium phosphate precipitation,polybrene mediated transfection, protoplast fusion, electroporation,encapsulation of the polynucleotide(s) in liposomes, mixing nucleic acidwith positively-charged lipids, and direct microinjection of the DNAinto nuclei.

Recombinant expression constructs of the invention typically comprise anucleic acid molecule encoding a polypeptide comprising one or more ofthe following: a heavy chain constant region (e.g., CH1, CH2 and/orCH3); a heavy chain variable region; a light chain constant region; alight chain variable region; one or more CDRs of the light or heavychain of the anti-DKK1 antibody. These nucleic acid sequences areinserted into an appropriate expression vector using standard ligationtechniques. In one embodiment, the 11H10 heavy or light chain constantregion is appended to the C-terminus of the DKK1-specific heavy or lightchain variable region and is ligated into an expression vector. Thevector is typically selected to be functional in the particular hostcell employed (i.e., the vector is compatible with the host cellmachinery, permitting amplification and/or expression of the gene canoccur). In some embodiments, vectors are used that employprotein-fragment complementation assays using protein reporters, such asdihydrofolate reductase (see, for example, U.S. Pat. No. 6,270,964).Suitable expression vectors can be purchased, for example, fromInvitrogen Life Technologies or BD Biosciences (formerly “Clontech”).Other useful vectors for cloning and expressing the antibodies andfragments of the invention include those described in Bianchi andMcGrew, Biotech Biotechnol Bioeng 84(4):439-44 (2003). Additionalsuitable expression vectors are discussed, for example, in MethodsEnzymol, vol. 185 (D. V. Goeddel, ed.), 1990, New York: Academic Press,which is hereby incorporated by reference.

Typically, expression vectors used in any of the host cells containsequences for plasmid or virus maintenance and for cloning andexpression of exogenous nucleotide sequences. Such sequences,collectively referred to as “flanking sequences” typically include oneor more of the following operatively linked nucleotide sequences: apromoter, one or more enhancer sequences, an origin of replication, atranscriptional termination sequence, a complete intron sequencecontaining a donor and acceptor splice site, a sequence encoding aleader sequence for polypeptide secretion, a ribosome binding site, apolyadenylation sequence, a polylinker region for inserting the nucleicacid encoding the polypeptide to be expressed, and a selectable markerelement.

Optionally, the vector may contain a “tag”-encoding sequence, that is,an oligonucleotide molecule located at the 5′ or 3′ end of the codingsequence, the oligonucleotide sequence encoding polyHis (such ashexaHis), or another “tag” for which commercially available antibodiesexist, such as FLAG©, HA (hemaglutinin from influenza virus), or myc.The tag is typically fused to the antibody protein upon expression, andcan serve as a means for affinity purification of the antibody from thehost cell. Affinity purification can be accomplished, for example, bycolumn chromatography using antibodies against the tag as an affinitymatrix. Optionally, the tag can subsequently be removed from thepurified antibody polypeptide by various means such as using certainpeptidases for cleavage.

Flanking sequences in the expression vector may be homologous (i.e.,from the same species and/or strain as the host cell), heterologous(i.e., from a species other than the host cell species or strain),hybrid (i.e., a combination of flanking sequences from more than onesource), synthetic or native. As such, the source of a flanking sequencemay be any prokaryotic or eukaryotic organism, any vertebrate orinvertebrate organism, or any plant, provided that the flanking sequenceis functional in, and can be activated by, the host cell machinery.

Flanking sequences useful in the vectors of this invention may beobtained by any of several methods well known in the art. Typically,flanking sequences useful herein will have been previously identified bymapping and/or by restriction endonuclease digestion and can thus beisolated from the proper tissue source using the appropriate restrictionendonucleases. In some cases, the full nucleotide sequence of a flankingsequence may be known. Here, the flanking sequence may be synthesizedusing the methods described herein for nucleic acid synthesis orcloning.

Where all or only a portion of the flanking sequence is known, it may beobtained using PCR and/or by screening a genomic library with a suitableoligonucleotide and/or flanking sequence fragment from the same oranother species. Where the flanking sequence is not known, a fragment ofDNA containing a flanking sequence may be isolated from a larger pieceof DNA that may contain, for example, a coding sequence or even anothergene or genes. Isolation may be accomplished by restriction endonucleasedigestion to produce the proper DNA fragment followed by isolation usingagarose gel purification, Qiagen™ column chromatography (Chatsworth,Calif.), or other methods known to the skilled artisan. The selection ofsuitable enzymes to accomplish this purpose will be readily apparent tothose skilled in the art.

An origin of replication is typically a part of prokaryotic expressionvectors, particularly those purchased commercially, and the origin aidsin the amplification of the vector in a host cell. If the vector ofchoice does not contain an origin of replication site, one may bechemically synthesized based on a known sequence, and ligated into thevector. For example, the origin of replication from the plasmid pBR322(New England Biolabs, Beverly, Mass.) is suitable for most gram-negativebacteria and various origins (e.g., SV40, polyoma, adenovirus, vesicularstomatitis virus (VSV), or papillomaviruses such as HPV or BPV) areuseful for cloning vectors in mammalian cells. Generally, a mammalianorigin of replication is not needed for mammalian expression vectors(for example, the SV40 origin is often used only because it contains theearly promoter).

The expression and cloning vectors of the present invention willtypically contain a promoter that is recognized by the host organism andoperably linked to nucleic acid encoding the anti-DKK1 antibody orimmunologically functional fragment thereof Promoters are untranscribedsequences located upstream (i.e., 5′) to the start codon of a structuralgene (generally within about 100 to 1000 bp) that control transcriptionof the structural gene. Promoters are conventionally grouped into one oftwo classes: inducible promoters and constitutive promoters. Induciblepromoters initiate increased levels of transcription from DNA undertheir control in response to some change in culture conditions, such asthe presence or absence of a nutrient or a change in temperature.Constitutive promoters, on the other hand, initiate continuous geneproduct production; that is, there is little or no experimental controlover gene expression. A large number of promoters, recognized by avariety of potential host cells, are well known. A suitable promoter isoperably linked to the DNA encoding anti-DKK1 antibody by removing thepromoter from the source DNA by restriction enzyme digestion oramplifying the promoter by polymerase chain reaction and inserting thedesired promoter sequence into the vector.

Suitable promoters for use with yeast hosts are also well known in theart. Yeast enhancers are advantageously used with yeast promoters.Suitable promoters for use with mammalian host cells are well known andinclude, but are not limited to, those obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, retroviruses, hepatitis-B virus and—most preferablySimian Virus 40 (SV40). Other suitable mammalian promoters includeheterologous mammalian promoters, for example, heat-shock promoters andthe actin promoter.

Particular promoters useful in the practice of the recombinantexpression vectors of the invention include, but are not limited to: theSV40 early promoter region (Bemoist and Chambon, 1981, Nature 290:304-10); the CMV promoter; the promoter contained in the 3′ longterminal repeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell 22:787-97); the herpes thymidine kinase promoter (Wagner et al., 1981,Proc. Natl. Acad. Sci. U.S.A. 78: 1444-45); the regulatory sequences ofthe metallothionine gene (Brinster et al., 1982, Nature 296: 39-42);prokaryotic expression vectors such as the beta-lactamase promoter(Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. U.S.A., 75:3727-31); or the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad.Sci. U.S.A. 80: 21-25). Also available for use are the following animaltranscriptional control regions, which exhibit tissue specificity andhave been utilized in transgenic animals: the elastase I gene controlregion that is active in pancreatic acinar cells (Swift et al., 1984,Cell 38: 63946; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant.Biol. 50: 399409; MacDonald, 1987, Hepatology 7: 425-515); the insulingene control region that is active in pancreatic beta cells (Hanahan,1985, Nature 315: 115-22); the mouse mammary tumor virus control regionthat is active in testicular, breast, lymphoid and mast cells (Leder etal., 1986, Cell 45: 485-95); the albumin gene control region that isactive in liver (Pinkert et al., 1987, Genes and Devel. 1: 268-76); thealpha-feto-protein gene control region that is active in liver (Krumlaufet al., 1985, Mol. Cell. Biol. 5: 1639-48; Hammer et al., 1987, Science235: 53-58); the alpha 1-antitrypsin gene control region that is activein the liver (Kelsey et al., 1987, Genes and Devel. 1: 161-71); thebeta-globin gene control region that is active in myeloid cells (Mogramet al., 1985, Nature 315: 338-40; Kollias et al., 1986, Cell 46: 89-94);the myelin basic protein gene control region that is active inoligodendrocyte cells in the brain (Readhead et al., 1987, Cell 48:703-12); the myosin light chain-2 gene control region that is active inskeletal muscle (Sani, 1985, Nature 314: 283-86); the gonadotropicreleasing hormone gene control region that is active in the hypothalamus(Mason et al., 1986, Science 234: 1372-78); and most particularly theimmunoglobulin gene control region that is active in lymphoid cells(Grosschedl et al., 1984, Cell 38: 647-58; Adames et al., 1985, Nature318: 533-38; Alexander et al., 1987, Mol. Cell Biol. 7: 1436-44).

An enhancer sequence may be inserted into the vector to increase thetranscription in higher eukaryotes of a nucleic acid encoding ananti-DKK1 antibody or immunologically functional fragment thereof of thepresent invention. Enhancers are cis-acting elements of DNA, usuallyabout 10-300 bp in length, that act on promoters to increasetranscription. Enhancers are relatively orientation and positionindependent. They have been found 5′ and 3′ to the transcription unit.Several enhancer sequences available from mammalian genes are known(e.g., globin, elastase, albumin, alpha-feto-protein and insulin). Anenhancer sequence from a virus also can be used. The SV40 enhancer, thecytomegalovirus early promoter enhancer, the polyoma enhancer, andadenovirus enhancers are exemplary enhancing elements for the activationof eukaryotic promoters. While an enhancer may be spliced into thevector at a position 5′ or 3′ to a nucleic acid molecule, it istypically placed at a site 5′ to the promoter.

In expression vectors, a transcription termination sequence is typicallylocated 3′ of the end of a polypeptide-coding region and serves toterminate transcription. A transcription termination sequence used forexpression in prokaryotic cells typically is a G-C rich fragmentfollowed by a poly-T sequence. While the sequence is easily cloned froma library or even purchased commercially as part of a vector, it canalso be readily synthesized using methods for nucleic acid synthesissuch as those described herein.

A selectable marker gene element encodes a protein necessary for thesurvival and growth of a host cell grown in a selective culture medium.Typical selection marker genes used in expression vectors encodeproteins that (a) confer resistance to antibiotics or other toxins,e.g., ampicillin, tetracycline, or kanamycin for prokaryotic host cells;(b) complement auxotrophic deficiencies of the cell; or (c) supplycritical nutrients not available from complex media. Examples ofselectable markers include the kanamycin resistance gene, the ampicillinresistance gene and the tetracycline resistance gene. A bacterialneomycin resistance gene can also be used for selection in bothprokaryotic and eukaryotic host cells.

Other selection genes can be used to amplify the gene that will beexpressed. Amplification is a process whereby genes that cannot insingle copy be expressed at high enough levels to permit survival andgrowth of cells under certain selection conditions are reiterated intandem within the chromosomes of successive generations of recombinantcells. Examples of suitable amplifiable selectable markers for mammaliancells include dihydrofolate reductase (DHFR) and promoterless thymidinekinase. In the use of these markers mammalian cell transformants areplaced under selection pressure wherein only the transformants areuniquely adapted to survive by virtue of the selection gene present inthe vector. Selection pressure is imposed by culturing the transformedcells under conditions in which the concentration of selection agent inthe medium is successively increased, thereby permitting survival ofonly those cells in which the selection gene has been amplified. Underthese circumstances, DNA adjacent to the selection gene, such as DNAencoding an antibody of the invention, is co-amplified with theselection gene. As a result, increased quantities of anti-DKK1polypeptide are synthesized from the amplified DNA.

A ribosome-binding site is usually necessary for translation initiationof mRNA and is characterized by a Shine-Dalgarno sequence (prokaryotes)or a Kozak sequence (eukaryotes). The element is typically located 3′ tothe promoter and 5′ to the coding sequence of the polypeptide to beexpressed.

In some cases, for example where glycosylation is desired in aeukaryotic host cell expression system, various presequences can bemanipulated to improve glycosylation or yield. For example, thepeptidase cleavage site of a particular signal peptide can be altered,or pro-sequences added, which also may affect glycosylation. The finalprotein product may have, in the −1 position (relative to the firstamino acid of the mature protein) one or more additional amino acidsincident to expression, which may not have been totally removed. Forexample, the final protein product may have one or two amino acidresidues found in the peptidase cleavage site, attached to theamino-terminus. Alternatively, use of some enzyme cleavage sites mayresult in a slightly truncated yet active form of the desiredpolypeptide, if the enzyme cuts at such area within the maturepolypeptide.

Where a commercially available expression vector lacks some of thedesired flanking sequences as described above, the vector can bemodified by individually ligating these sequences into the vector. Afterthe vector has been chosen and modified as desired, a nucleic acidmolecule encoding an anti-DKK1 antibody or immunologically functionalfragment thereof is inserted into the proper site of the vector.

The completed vector containing sequences encoding the inventiveantibody or immunologically functional fragment thereof is inserted intoa suitable host cell for amplification and/or polypeptide expression.The transformation of an expression vector for an anti-DKK1 antibodyimmunologically functional fragment thereof into a selected host cellmay be accomplished by well-known methods including methods such astransfection, infection, calcium chloride, electroporation,microinjection, lipofection, DEAE-dextran method, or other knowntechniques. The method selected will in part be a function of the typeof host cell to be used. These methods and other suitable methods arewell known to the skilled artisan.

The transformed host cell, when cultured under appropriate conditions,synthesizes an anti-DKK1 antibody or functional fragment thereof thatcan subsequently be collected from the culture medium (if the host cellsecretes it into the medium) or directly from the host cell producing it(if it is not secreted). The selection of an appropriate host cell willdepend upon various factors, such as desired expression levels,polypeptide modifications that are desirable or necessary for activity(such as glycosylation or phosphorylation) and ease of folding into abiologically active molecule.

Mammalian cell lines available as hosts for expression are well known inthe art and include, but are not limited to, many immortalized celllines available from the American Type Culture Collection (ATCC), suchas Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney(BHK) cells, monkey kidney cells (COS), human hepatocellular carcinomacells (e.g., Hep G2), and a number of other cell lines. In certainembodiments, the best cell line for expressing a particular DNAconstruct may be selected by testing various cell lines to determinewhich ones have the highest levels of expression levels and produceantibodies with constitutive DKK1 binding properties.

In certain embodiments, the invention also provides compositionscomprising the subject anti-DKK1 antibodies or immunologicallyfunctional fragments thereof together with one or more of the following:a pharmaceutically acceptable diluent; a carrier; a solubilizer; anemulsifier; a preservative; and/or an adjuvant. Such compositions maycontain an effective amount of the anti-DKK1 antibody or immunologicallyfunctional fragment thereof. Thus, the use of the antibodies andimmunologically active fragments that are provided herein in thepreparation of a pharmaceutical composition or medicament is alsoincluded. Such compositions can be used in the treatment of a variety ofdiseases such as listed below in the section on exemplary utilities.

Acceptable formulation components for pharmaceutical preparations arenontoxic to recipients at the dosages and concentrations employed. Inaddition to the antibodies and immunologically functional fragments thatare provided, compositions according to the invention may containcomponents for modifying, maintaining or preserving, for example, thepH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility,stability, rate of dissolution or release, adsorption or penetration ofthe composition. Suitable materials for formulating pharmaceuticalcompositions include, but are not limited to, amino acids (such asglycine, glutamine, asparagine, arginine or lysine); antimicrobials;antioxidants (such as ascorbic acid, sodium sulfite or sodiumhydrogen-sulfite); buffers (such as acetate, borate, bicarbonate,Tris-HCl, citrates, phosphates or other organic acids); bulking agents(such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine,polyvinylpyrrolidone, beta-cyclodextrin orhydroxypropyl-beta-cyclodextrin); fillers; monosaccharides;disaccharides; and other carbohydrates (such as glucose, mannose ordextrins); proteins (such as serum albumin, gelatin or immunoglobulins);coloring, flavoring and diluting agents; emulsifying agents; hydrophilicpolymers (such as polyvinylpyrrolidone); low molecular weightpolypeptides; salt-forming counterions (such as sodium); preservatives(such as benzalkonium chloride, benzoic acid, salicylic acid,thimerosal, phenethyl alcohol, methylparaben, propylparaben,chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such asglycerin, propylene glycol or polyethylene glycol); sugar alcohols (suchas mannitol or sorbitol); suspending agents; surfactants or wettingagents (such as pluronics, PEG, sorbitan esters, polysorbates such aspolysorbate 20, polysorbate 80, triton, tromethamine, lecithin,cholesterol, tyloxapal); stability enhancing agents (such as sucrose orsorbitol); tonicity enhancing agents (such as alkali metal halides,preferably sodium or potassium chloride, mannitol sorbitol); deliveryvehicles; diluents; excipients and/or pharmaceutical adjuvants. (seeRemington's Pharmaceutical Sciences, 18th Edition, (A. R. Gennaro, ed.),1990, Mack Publishing Company), hereby incorporated by reference.

The primary vehicle or carrier in a pharmaceutical composition may beeither aqueous or non-aqueous in nature. Suitable vehicles or carriersfor such compositions include water for injection, physiological salinesolution or artificial cerebrospinal fluid, possibly supplemented withother materials common in compositions for parenteral administration.Neutral buffered saline or saline mixed with serum albumin are furtherexemplary vehicles. Compositions comprising anti-DKK1 antibodies orimmunologically functional fragments thereof may be prepared for storageby mixing the selected composition having the desired degree of puritywith optional formulation agents in the form of a lyophilized cake or anaqueous solution. Further, the anti-DKK1 antibodies or immunologicallyfunctional fragments thereof may be formulated as a lyophilizate usingappropriate excipients such as sucrose.

Formulation components are present in concentrations that are acceptableto the site of administration. Buffers are advantageously used tomaintain the composition at physiological pH or at a slightly lower pH,typically within a pH range of from about 4.0 to about 8.5, oralternatively, between about 5.0 to 8.0. Pharmaceutical compositions cancomprise TRIS buffer of about pH 6.5-8.5, or acetate buffer of about pH4.0-5.5, which may further include sorbitol or a suitable substitutetherefor.

A pharmaceutical composition may involve an effective quantity ofanti-DKK1 antibodies or immunologically functional fragments thereof ina mixture with non-toxic excipients that are suitable for themanufacture of tablets. By dissolving the tablets in sterile water, oranother appropriate vehicle, solutions may be prepared in unit-doseform. Suitable excipients include, but are not limited to, inertmaterials, such as calcium carbonate, sodium carbonate or bicarbonate,lactose, or calcium phosphate; or binding agents, such as starch,gelatin, or acacia; or lubricating agents such as magnesium stearate,stearic acid, or talc.

Additional pharmaceutical compositions are in the form of sustained- orcontrolled-delivery formulations. Techniques for formulating a varietyof other sustained- or controlled-delivery means, such as liposomecarriers, bio-erodible microparticles or porous beads and depotinjections can be used (see, for e.g., PCT/US93/00829, which describesthe controlled release of porous polymeric microparticles for thedelivery of pharmaceutical compositions). Sustained-release preparationsmay include semipermeable polymer matrices in the form of shapedarticles, e.g. films, or microcapsules, polyesters, hydrogels,polylactides (U.S. Pat. No. 3,773,919 and EP 058,481), copolymers ofL-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., 1983,Biopolymers 22: 547-556), poly (2-hydroxyethyl-methacrylate) (Langer etal., 1981, J Biomed Mater Res 15: 167-277) and Langer, 1982, Chem Tech12: 98-105), ethylene vinyl acetate (Langer et al., ibid.) orpoly-D(−)-3-hydroxybutyric acid (EP 133,988). Sustained releasecompositions may also include liposomes, which can be prepared by any ofseveral methods known in the art. See e.g., Eppstein et al., 1985, Proc.Natl. Acad. Sci. USA 82: 3688-3692; EP 036,676; EP 088,046 and EP143,949.

The pharmaceutical composition to be used for in vivo administrationtypically is sterile. Sterilization may be accomplished by filtrationthrough sterile filtration membranes. If the composition is lyophilized,sterilization may be conducted either prior to or followinglyophilization and reconstitution. The composition for parenteraladministration may be stored in lyophilized form or in a solution. Incertain embodiments, parenteral compositions are placed into a containerhaving a sterile access port, for example, an intravenous solution bagor vial having a stopper pierceable by a hypodermic injection needle, ora sterile pre-filled syringe ready to use for injection.

Once the pharmaceutical composition of the invention has beenformulated, it may be stored in sterile vials as a solution, suspension,gel, emulsion, solid, or as a dehydrated or lyophilized powder. Suchformulations may be stored either in a ready-to-use form or in a form(e.g., lyophilized) that is reconstituted prior to administration.

The components used to formulate the pharmaceutical compositions arepreferably of high purity and are substantially free of potentiallyharmful contaminants (e.g., at least National Food (NF) grade, generallyat least analytical grade, and more typically at least pharmaceuticalgrade). Moreover, compositions intended for in vivo use are usuallysterile. To the extent that a given compound must be synthesized priorto use, the resulting product is typically substantially free of anypotentially toxic agents, particularly any endotoxins, which may bepresent during the synthesis or purification process. Compositions forparental administration are also sterile, substantially isotonic andmade under GMP conditions.

The present invention provides kits for producing a multi-dose orsingle-dose administration units. For example, kits according to theinvention may each contain both a first container having a dried proteinand a second container having an aqueous diluent, including for examplesingle and multi-chambered pre-filled syringes (e.g., liquid syringes,lyosyringes or needle-free syringes).

The pharmaceutical compositions of the invention can be deliveredparenterally, typically by injection. Injections can be intraocular,intraperitoneal, intraportal, intramuscular, intravenous, intrathecal,intracerebral (intra-parenchymal), intracerebroventricular,intraarterial, intralesional, perilesional or subcutaneous. Eye dropscan be used for intraocular administration. In some instances,injections may be localized to the vicinity of a particular bone orbones to which the treatment is targeted. For parenteral administration,the antibodies may be administered in a pyrogen-free, parenterallyacceptable aqueous solution comprising the desired anti-DKK1 antibodiesor immunologically functional fragments thereof in a pharmaceuticallyacceptable vehicle. A particularly suitable vehicle for parenteralinjection is sterile distilled water in which the anti-DKK1 antibodiesor immunologically functional fragments thereof are formulated as asterile, isotonic solution, properly preserved.

Pharmaceutical compositions comprising the subject anti-DKK1 antibodiesand functional fragments thereof may be administered by bolus injectionor continuously by infusion, by implantation device, sustained releasesystems or other means for accomplishing prolonged release. Thepharmaceutical composition also can be administered locally viaimplantation of a membrane, sponge or another appropriate material ontowhich the desired molecule has been absorbed or encapsulated. Where animplantation device is used, the device may be implanted into anysuitable tissue or organ, and delivery of the desired molecule may bevia diffusion, timed-release bolus, or continuous release. Thepreparation may be formulated with agent, such as injectablemicrospheres, bio-erodible particles, polymeric compounds (such aspolylactic acid; polyglycolic acid; or copoly (lactic/glycolic) acid(PLGA), beads or liposomes, that can provide controlled or sustainedrelease of the product which may then be delivered via a depotinjection. Formulation with hyaluronic acid has the effect of promotingsustained duration in the circulation.

The subject compositions comprising an anti-DKK1 antibody or functionalfragment thereof may be formulated for inhalation. In these embodiments,an anti-DKK1 antibody is formulated as a dry powder for inhalation, oranti-DKK1 antibody inhalation solutions may also be formulated with apropellant for aerosol delivery, such as by nebulization. Pulmonaryadministration is further described in PCT/US94/001875, which describespulmonary delivery of chemically modified proteins, and which is herebyincorporated by reference.

Certain pharmaceutical compositions of the invention can be deliveredthrough the digestive tract, such as orally. The subject anti-DKK1antibodies or immunologically functional fragments thereof that areadministered in this fashion may be formulated with or without thosecarriers customarily used in the compounding of solid dosage forms suchas tablets and capsules. A capsule may be designed to release the activeportion of the formulation at the point in the gastrointestinal tractwhen bioavailability is maximized and pre-systemic degradation isminimized. Additional agents can be included to facilitate absorption ofthe anti-DKK1 antibody or functional fragment thereof. For oraladministration, modified amino acids may be used to confer resistance todigestive enzymes. Diluents, flavorings, low melting point waxes,vegetable oils, lubricants, suspending agents, tablet disintegratingagents, and binders may also be employed.

The subject compositions comprising anti-DKK1 antibodies orimmunologically functional fragments thereof also may be used ex vivo.In such instances, cells, tissues or organs that have been removed fromthe patient are exposed to or cultured with the anti-DKK1 antibody. Thecultured cells may then be implanted back into the patient or adifferent patient or used for other purposes.

In certain embodiments, anti-DKK1 antibodies or immunologicallyfunctional fragments thereof can be delivered by implanting certaincells that have been genetically engineered, using methods such as thosedescribed herein, to express and secrete the polypeptide. Such cells maybe animal or human cells, and may be autologous, heterologous, orxenogenic, or may be immortalized. In order to decrease the chance of animmunological response, the cells may be encapsulated to avoidinfiltration of surrounding tissues. Encapsulation materials aretypically biocompatible, semi-permeable polymeric enclosures ormembranes that allow the release of the protein product(s) but preventthe destruction of the cells by the patient's immune system or by otherdetrimental factors from the surrounding tissues.

Dosage

The pharmaceutical compositions that are provided can be administeredfor prophylactic and/or therapeutic treatments. An “effective amount”refers generally to an amount that is a sufficient, but non-toxic,amount of the active ingredient (i.e., an anti-DKK1 antibody orimmunologically functional fragment thereof) to achieve the desiredeffect, which is a reduction or elimination in the severity and/orfrequency of symptoms and/or improvement or remediation of damage. A“therapeutically effective amount” refers to an amount that issufficient to remedy a disease state or symptoms, or otherwise prevent,hinder, retard or reverse the progression of a disease or any otherundesirable symptom. A “prophylactically effective amount” refers to anamount that is effective to prevent, hinder or retard the onset of adisease state or symptom.

In general, toxicity and therapeutic efficacy of the antibody orfragment can be determined according to standard pharmaceuticalprocedures in cell cultures and/or experimental animals, including, forexample, determining the LD50 (the dose lethal to 50% of the population)and the ED50 (the dose therapeutically effective in 50% of thepopulation). The dose ratio between toxic and therapeutic effects is thetherapeutic index and it can be expressed as the ratio LD50/ED50.Compositions that exhibit large therapeutic indices are preferred.

The data obtained from cell culture and/or animal studies can be used informulating a range of dosages for humans. The dosage of the activeingredient typically lines within a range of circulating concentrationsthat include the ED50 with little or no toxicity. The dosage can varywithin this range depending upon the dosage form employed and the routeof administration utilized.

The effective amount of a pharmaceutical composition comprisinganti-DKK1 antibodies or immunologically functional fragments thereof tobe employed therapeutically or prophylactically will depend, forexample, upon the therapeutic context and objectives. One skilled in theart will appreciate that the appropriate dosage levels for treatment,according to certain embodiments, will thus vary depending, in part,upon the molecule delivered, the indication for which the anti-DKK1antibody is being used, the route of administration, and the size (bodyweight, body surface or organ size) and/or condition (the age andgeneral health) of the patient. A clinician may titer the dosage andmodify the route of administration to obtain the optimal therapeuticeffect. Typical dosages range from about 0.1 μg/kg to up to about 100mg/kg or more, depending on the factors mentioned above. In certainembodiments, the dosage may range from 0.1 μg/kg up to about 150 mg/kg;or 1 μg/kg up to about 100 mg/kg; or 5 μg/kg up to about 50 mg/kg.

The dosing frequency will depend upon the pharmacokinetic parameters ofthe anti-DKK1 antibody or immunologically functional fragment thereof inthe formulation. For example, a clinician will administer thecomposition until a dosage is reached that achieves the desired effect.The composition may therefore be administered as a single dose, or astwo or more doses (which may or may not contain the same amount of thedesired molecule) over time, or as a continuous infusion via animplantation device or catheter. Treatment may be continuous over timeor intermittent. Further refinement of the appropriate dosage isroutinely made by those of ordinary skill in the art and is within theambit of tasks routinely performed by them. Appropriate dosages may beascertained through use of appropriate dose-response data.

To treat a medical disorder by targeting DKK1, a composition comprisingthe subject anti-DKK1 antibodies or immunologically functional fragmentsthereof may be administered to the patient in an amount and for a timesufficient to induce a sustained improvement in at least one indicatorthat reflects the severity of the disorder. An improvement is considered“sustained” if the patient exhibits the improvement on at least twooccasions separated by at least one to seven days, or in some instancesone to six weeks. The appropriate interval will depend to some extent onwhat disease condition is being treated; it is within the purview of theskilled physician to determine the appropriate interval for determiningwhether the improvement is sustained. The degree of improvement isdetermined based on signs or symptoms, and may also employquestionnaires that are administered to the patient, such asquality-of-life questionnaires.

Various indicators that reflect the extent of the patient's illness maybe assessed for determining whether the amount and time of the treatmentis sufficient. The baseline value for the chosen indicator or indicatorsis established by examination of the patient prior to administration ofthe first dose of antibody. Preferably, the baseline examination is donewithin about 60 days of administering the first dose. If the antibody isbeing administered to treat acute symptoms, such as for example to treata broken bone, the first dose is administered as soon as practicallypossible after the injury has occurred.

Improvement is induced by administering the subject anti-DKK1 antibodiesor immunologically functional fragments thereof until the patientmanifests an improvement over baseline for the chosen indicator orindicators. In treating chronic conditions, this degree of improvementis obtained by repeatedly administering this medicament over a period ofat least a month or more, e.g., for one, two, or three months or longer,or indefinitely. A period of one to six weeks, or even a single dose,often is sufficient for treating acute conditions. For injuries or acuteconditions, a single dose may be sufficient.

Although the extent of the patient's illness after treatment may appearimproved according to one or more indicators, treatment may be continuedindefinitely at the same level or at a reduced dose or frequency. Oncetreatment has been reduced or discontinued, it later may be resumed atthe original level if symptoms should reappear.

The subject anti-DKK1 antibodies and immunologically functionalfragments thereof can be used to detect DKK1 in biological samples. Suchuses allow the identification of cells or tissues that produce theprotein or serve as a diagnostic for detecting pathological conditionsin which DKK1 is overproduced or underproduced. The antibodies andfragments that are provided can also be used in methods to screen for amolecule that binds to DKK1. A variety of competitive screening methods,for example, can be used. In some methods, a DKK1 molecule or fragmentthereof to which an anti-DKK1 antibody binds, is contacted with anantibody or fragment disclosed herein together with another molecule(i.e., a candidate molecule). A reduction in binding between theantibody or fragment and DKK1 is an indication that the molecule bindsDKK1. Binding of the antibody or fragment can be detected using avariety of methods, e.g., an ELISA. Detection of binding between theanti-DKK1 antibody or fragment to DKK1 can be simplified by detectablylabeling the antibody. In some methods, a molecule that exhibits bindingin the initial screen is further analyzed to determine whether itinhibits a DKK1 activity (e.g., whether the molecule activates Wntsignaling).

Activity of a DKK1 inhibitor or a sclerostin inhibitor or combinations(e.g., respective binding agents) may be measured in a variety of ways.Binding agent-mediated increases in bone mineral content or bone densitymay be measured using single- and dual-energy X-ray absorptometry,ultrasound, computed tomography, radiography, and magnetic resonanceimaging. The amount of bone mass may also be calculated from bodyweights or by using other methods (see Guinness-Hey, Metab. Bone Dis.Relat. Res., 5:177-181 (1984)). Animals and particular animal models areused in the art for testing the effect of the pharmaceuticalcompositions and methods on, for example, parameters of bone loss, boneresorption, bone formation, bone strength, or bone mineralization thatmimic conditions of human disease such as osteoporosis and osteopenia.Examples of such models include the ovariectomized rat model (Kalu, Boneand Mineral, 15:175-192 (1991); Frost and Jee, Bone and Mineral,18:227-236 (1992); and Jee and Yao, J. Musculoskel. Neuron. Interact.,1:193-207 (2001)). The methods for measuring binding agent activitydescribed herein also may be used to determine the efficacy of otherinhibitors.

In humans, bone mineral density can be determined clinically using dualx-ray absorptiometry (DXA) of, for example, the hip and spine. Othertechniques include quantitative computed tomography (QCT),ultrasonography, single-energy x-ray absorptiometry (SXA), andradiographic absorptiometry. Common central skeletal sites formeasurement include the spine and hip; peripheral sites include theforearm, finger, wrist and heel. Except for ultrasonography, theAmerican Medical Association notes that BMD techniques typically involvethe use of x-rays and are based on the principle that attenuation of theradiation depends on thickness and composition of the tissues in theradiation path. All techniques involve the comparison of results to anormative database.

Alternatively, a physiological response to one or more binding agentscan be gauged by monitoring bone marker levels. Bone markers areproducts created during the bone remodeling process and are released bybone, osteoblasts, and/or osteoclasts. Fluctuations in bone resorptionand/or bone formation “marker” levels imply changes in boneremodeling/modeling. The International Osteoporosis Foundation (IOF)recommends using bone markers to monitor bone density therapies (see,e.g., Delmas et al., Osteoporos Int., Suppl. 6:S2-17 (2000),incorporated herein by reference). Markers indicative of bone resorption(or osteoclast activity) include, for example, C-telopeptide (e.g.,C-terminal telopeptide of type 1 collagen (CTX) or serum cross-linkedC-telopeptide), N-telopeptide (N-terminal telopeptide of type 1 collagen(NTX)), deoxypyridinoline (DPD), pyridinoline, urinary hydroxyproline,galactosyl hydroxylysine, and tartrate-resistant acid phosphatase (e.g.,serum tartrate-resistant acid phosphatase isoform 5b). Boneformation/mineralization markers include, but are not limited to,bone-specific alkaline phosphatase (BSAP), peptides released from N- andC-terminal extension of type I procollagen (P1NP, PICP), and osteocalcin(OstCa). Several kits are commercially-available to detect and quantifymarkers in clinical samples, such as urine and blood.

Upon administration, a therapeutic agent preferably reduces the level ofone or more markers of bone resorption, such as the serum level ofC-telopeptide of type I collagen (CTX). Accordingly, the inventionfurther provides a method of monitoring therapy, i.e., the physiologicalresponse to a sclerostin binding agent or other sclerostin inhibitor.The method comprises administering a therapeutic, then measuring thelevel of one or more markers of bone resorption. In addition, the methodcan comprise measuring the level of one or more markers of boneformation before administration. The level of bone resorption markerduring and/or after treatment may be compared to a pre-treatment level,or alternatively may be compared to a standard range typical of thatpatient population. One of ordinary skill in the art can readilydetermine a suitable standard range by testing a representative numberof patients of like age, gender, disease level, and/or othercharacteristics of the patient population. The level of bone resorptionmarker can be reduced by at least about 5% (e.g., about 10%, about 20%,or about 30%) by a single dose of therapeutic. In some embodiments, thedose of therapeutic reduces the level of bone resorption marker at leastabout 40% (e.g., about 50%, about 60%, or about 70%) compared to thelevel of the bone resorption marker prior to administering. In addition,the bone resorption marker level may be reduced for at least about 3days (e.g., about 7 days, about 2 weeks, about 3 weeks, about 1 month,about 5 weeks, about 6 weeks, about 7 weeks, about 2 months, about 9weeks, about 10 weeks, about 11 weeks, or about 3 months) afteradministration of a single dose.

In addition to decreasing the level of bone resorption markers, theamount of therapeutic administered to a patient also can increase thelevel of one or more markers of bone formation, such as the serum levelof BSAP, the serum level of P1NP, and/or the serum level of OstCa. Asingle dose of therapeutic can increase the level of a bone formationmarker by, for example, at least about 5% (e.g., about 10%, about 20%,or about 30%). In some embodiments, the dose of therapeutic elevates thelevel of a bone formation marker at least about 40% (e.g., about 50%,about 60%, or about 70%). In other embodiments, the dose of therapeuticincreases the level of one or more bone formation markers by at leastabout 75% (e.g., about 80%, about 90%, about 100%, or about 110%). Inyet other embodiments, the dose of therapeutic increases the level of abone formation marker by at least about 120% (e.g., about 130%, about140%, about 150%, about 160% or about 170%). In alternative embodiments,the therapeutic increases the level of bone formation marker by leastabout 180% (e.g., about 190% or about 200%). Bone formation markerlevels ideally remain elevated (compared to bone formation marker levelspre-treatment or to a standard range typical of that patient population)for at least about 3 days (e.g., about 7 days, about 2 weeks, about 3weeks, about 1 month, about 5 weeks, about 6 weeks, about 7 weeks, about2 months, about 9 weeks, about 10 weeks, about 11 weeks, or about 3months) after administration of a single dose of the therapeutic.

Typically, BMD is can be measured “total body” (e.g., head, trunk, arms,and legs) or at the hip (e.g., total hip and/or femoral neck), spine(e.g., lumbar spine), wrist, finger, shin bone and/or heel. Inosteoporosis diagnosis, a patient's BMD is compared to the peak densityof a 30-year old healthy adult (i.e., a “young adult”), creating theso-called “T-score.” A patient's BMD also may be compared to an“age-matched” bone density (see, e.g., World Health OrganizationScientific Group on the Prevention and Management of Osteoporosis,“Prevention and management of osteoporosis: report of a WHO scientificgroup.” WHO Technical Report Series; 921, Geneva, Switzerland (2000)).The difference between a patient's BMD and that of a healthy, youngadult is conventionally referred to in terms of the multiple of a“standard deviation,” which typically equals about 10% to about 12%decrease in bone density. The World Health Organization proposed fourdiagnostic categories based on BMD T-scores. A BMD value within 1standard deviation of the young adult reference mean (T-score≤−1) is“normal.” Low bone mass (osteopenia) is indicated by a BMD value morethan 1 standard deviation below the young adult mean, but less than 2standard deviations (T-score<−1 and >−2.5). A T-score of more than 2.5standard deviations below the norm supports a diagnosis of osteoporosis.If a patient additionally suffers from one or more fragility fractures,the patient qualifies as having severe osteoporosis.

The therapeutic may be administered to a patient to improve bone mineraldensity regardless of the patient's T-score. The therapeutic may beadministered at a dose and for a time period effective to increase BMDin the patient by at least about 1% (about 2%, about 3%, about 4%, about5%, or about 6%). In some embodiments, BMD is increased by at leastabout 8% (e.g., at least about 10%, about 12%, about 15%, or about 18%).In other embodiments, BMD is increased by the therapeutic at least about20% (e.g., at least about 22%, about 25%, or about 28%) at the hip,spine, wrist, finger, shin bone, and/or heel. In yet other embodiments,BMD is increased at least about 30% (e.g., at least about 32%, about35%, about 38%, or about 40%). In other words, the BMD can be increasedto the range of about 1 to about 2.5 standard deviations (preferably arange of about 0 to about 1 standard deviations) below the normal BMD ofa healthy young adult.

Alterations in bone remodeling or modeling can lead to fluctuations inmineral concentrations throughout the body. Bone is one of the principalregulators of calcium levels in the bloodstream. Osteoclast-mediatedbone resorption releases stored calcium into the systemic circulation,while osteoblast-mediated bone formation removes calcium fromcirculation to incorporate into bone tissue. In normal bonemodeling/remodeling, these processes cycle to maintain healthy, strongbone and maintain free calcium levels at about 8.5 mg/dL to about 10.5mg/dL (e.g., about 2.2 mmol/L to about 2.6 mmol/L). Bone disorders,other illnesses, and even certain therapies can disrupt systemic calciumlevels with dire consequences. Hypercalcemia is associated with highlevels of calcium in the blood (e.g., greater than 12 mg/dL or 3mmol/L). Extraordinarily high calcium levels leads to, for example,fatigue, confusion, constipation, decreased appetite, frequenturination, heart problems, and bone pain. Hypocalcemia is an electrolyteimbalance indicated by an abnormally low level of calcium in the blood(e.g., less than about 9 mg/dL or 2.2 mmol/L). Calcium levels of <7.5mg/dL (<1.87 mmol/L) or less are considered severe hypocalcemia and maybe accompanied by clinical symptoms.

Methods of Treatment and Uses

The inventive methods are useful for treating or preventing bone-relateddisorders, such as bone-related disorders associated with abnormalosteoblast or osteoclast activity. Indeed, the therapeutics of thepresent invention can be administered to a human suffering from a bonerelated disorder selected from the group consisting of achondroplasia,cleidocranial dysostosis, enchondromatosis, fibrous dysplasia, Gaucher'sDisease, hypophosphatemia, X-linked hypophosphatemic rickets, Marfan'ssyndrome, multiple hereditary exotoses, neurofibromatosis, osteogenesisimperfecta, osteopetrosis, osteopoikilosis, sclerotic lesions,pseudoarthrosis, pyogenic osteomyelitis, periodontal disease,anti-epileptic drug induced bone loss, primary and secondaryhyperparathyroidism, familial hyperparathyroidism syndromes,weightlessness induced bone loss, osteoporosis in men, postmenopausalbone loss, spinal fusion, osteoarthritis, renal osteodystrophy,infiltrative disorders of bone, oral bone loss, osteonecrosis of thejaw, juvenile Paget's disease, melorheostosis, metabolic bone diseases,mastocytosis, sickle cell anemia/disease, organ transplant related boneloss, kidney transplant related bone loss, systemic lupus erythematosus,ankylosing spondylitis, epilepsy, juvenile arthritides, thalassemia,mucopolysaccharidoses, Fabry Disease, Turner Syndrome, Down Syndrome,Klinefelter Syndrome, leprosy, Perthe's Disease, adolescent idiopathicscoliosis, infantile onset multi-system inflammatory disease, WinchesterSyndrome, Menkes Disease, Wilson's Disease, ischemic bone disease (suchas Legg-Calve-Perthes disease and regional migratory osteoporosis),anemic states, conditions caused by steroids, glucocorticoid-inducedbone loss, heparin-induced bone loss, bone marrow disorders, scurvy,malnutrition, calcium deficiency, osteoporosis, osteopenia, alcoholism,chronic liver disease, postmenopausal state, chronic inflammatoryconditions, rheumatoid arthritis, inflammatory bowel disease, ulcerativecolitis, inflammatory colitis, Crohn's disease, oligomenorrhea,amenorrhea, pregnancy, diabetes mellitus, hyperthyroidism, thyroiddisorders, parathyroid disorders, Cushing's disease, acromegaly,hypogonadism, immobilization or disuse, reflex sympathetic dystrophysyndrome, regional osteoporosis, osteomalacia, bone loss associated withjoint replacement, HIV associated bone loss, bone loss associated withloss of growth hormone, bone loss associated with cystic fibrosis,chemotherapy-associated bone loss, tumor-induced bone loss,cancer-related bone loss, hormone ablative bone loss, multiple myeloma,drug-induced bone loss, anorexia nervosa, disease-associated facial boneloss, disease-associated cranial bone loss, disease-associated bone lossof the jaw, disease-associated bone loss of the skull, bone lossassociated with aging, facial bone loss associated with aging, cranialbone loss associated with aging, jaw bone loss associated with aging,skull bone loss associated with aging, and bone loss associated withspace travel.

The inventive methods need not cure the patient of the disorder orcompletely protect against the onset of a bone-related disorder toachieve a beneficial biological response. The methods may be usedprophylactically, meaning to protect, in whole or in part, against abone-related disorder or symptom thereof. The methods also may be usedtherapeutically to ameliorate, in whole or in part, a bone-relateddisorder or symptom thereof, or to protect, in whole or in part, againstfurther progression of a bone-related disorder or symptom thereof.Indeed, the materials and methods of the invention are particularlyuseful for increasing bone mineral density and maintaining the increasedBMD over a period of time. In this regard, the invention provides amethod of treating a bone-related disorder, which method comprises (a)administering one or more amounts of a sclerostin binding agenteffective to increase BMD measured for the total body (e.g., head,trunk, arms, and legs) or at the hip (e.g., total hip and/or femoralneck), spine (e.g., lumbar spine), wrist, finger, shin bone and/or heelby about 1%, about 2%, about 3%, about 6%, about 8%, about 10%, about12%, about 15%, about 18%, about 20%, about 25%, or 30% or more. One ormore administrations of a pharmaceutical composition comprising thesclerostin binding agent may be carried out over a therapeutic periodof, for example, about 1 month to about 18 months (e.g., about 2 months,about 3 months, about 4 months, about 5 months, about 6 months, about 7months, about 8 months, about 9 months, about 10 months, or about 11months). The method further includes (b) subsequently administering oneor more amounts of a sclerostin binding agent effective to maintain bonemineral density. By “maintain bone mineral density” is meant that theincreased BMD resulting from step (a) does not fall more than about 1%to about 5% over the course of step (b) (e.g., about 6 months, about 9months about 1 year, about 18 months, about 2 years, or over the courseof the patient's life). It will be appreciated that a patient canrequire alternate treatment phases for increasing bone density andmaintaining bone density.

It is contemplated that the therapeutic use of DKK1 inhibitors, asdescribed herein, alone or in combination with another anabolic agent,e.g., a sclerostin inhibitor such as a neutralizing antibody, isbeneficial to any condition requiring bone repair whether it isaggravated by an underlying bone loss condition or not. Particularexamples of bone repair that are not always associated with bone lossinclude fracture repair such as delayed healing or non-union healing.Thus, one of skill in the art will understand that certain indicationsdescribed herein may or may not be exacerbated by bone loss associatedwith, for example, osteoporosis, or any other bone loss conditiondescribed herein. Thus in further embodiments it is contemplated thatcompositions of the present invention are useful for improving outcomesin orthopedic procedures, periodontal diseases, oral bone loss, dentalprocedures, dental implants, implant surgery, joint replacement, bonegrafting, bone cosmetic surgery and bone repair such as fracturehealing, spinal fusion, implant fixation (e.g., joint replacement suchas hip or knew), non-union healing, delayed union healing and facialreconstruction. One or more compositions may be administered before,during and/or after the procedure, replacement, graft, surgery orrepair.

In another aspect, the use of the foregoing therapeutic modalitiesincluding antibodies or immunologically functional fragments in thetreatment of a variety of diseases is disclosed. Certain methods, forinstance, involve administering to a patient in need thereof aneffective amount of an antibody or immunologically active fragment asdescribed herein to treat arthritis, diseases responsive to stem cellrenewal, inflammatory diseases, neurological diseases, ocular diseases,renal diseases, pulmonary diseases, and skin diseases. Some treatmentmethods involve treating rheumatoid arthritis, psoriatic arthritis orosteoarthritis. Certain antibodies and fragments are used to treat adisease that: (a) is responsive to stem cell renewal and is selectedfrom the group consisting of diabetes, chronic heart failure anddiseases of the muscle; (b) is an inflammatory disease selected from thegroup consisting of Crohn's disease, colitis, and inflammatory boweldisease; (c) is a neurological disease selected from the groupconsisting of Alzheimer's disease, Parkinson's disease, and Huntington'sdisease; (d) is an ocular disease selected from the group consisting ofmacular degeneration and retinopathies; (e) is a renal disease selectedfrom the group consisting of end stage renal disease, chronic renaldisease, glomerulonephritis, tubulointerstitial nephritis, and IgAnephropathy; (f) is a pulmonary disease selected from the groupconsisting of chronic obstructive pulmonary disease, idiopathicpulmonary fibrosis, and cystic fibrosis; or (g) is a skin diseaseresulting from chemotherapy-induced damage to the intestinal epithelium.

Sclerostin inhibitors, e.g., sclerostin binding agents, have been shownto promote bone formation and inhibit (or slow) bone resorption withminimal fluctuations in systemic calcium levels (e.g., calcium levelsfluctuate 10% or less from baseline serum calcium levels). Thus itpresents itself as a possible partner therapeutic with the DKK1inhibitors presented herein to increase therapeutic responsiveness.

Several ailments and pharmaceutical therapies alter system calciumlevels, and thereby impact bone density in a negative way and as suchtherapeutics of the current invention are useful, including combinationsthereof, to treat bone loss in these conditions. Hypercalcemia andhypocalcemia can result from, for example, chronic kidney disease, renaldiseases, renal failure, primary or secondary hyperparathyroidism,pseudohyperparathyroidism, hypoparathyroidism, pseudohypoparathyroidism,magnesium depletion, alcoholism, bisphosphonate therapy, severehypermagnesemia, vitamin D deficiency, hyperphosphatemia, acutepancreatitis, hungry bone syndrome, chelation, osteoblastic metastases,sepsis, surgery, chemotherapy, neoplasia syndrome, familialhypocalciuric hypercalcemia, sarcoidosis, tuberculosis, berylliosis,histoplasmosis, Candidiasis, Coccidioidomycosis, histiocytosis X,Hodgkin's or Non-Hodgkin's lymphoma, Crohn's disease, Wegener'sgranulomatosis, leukemia, pneumonia, silicone-induced granulomas,immobilization, or drug therapy, such as administration of thiazidediuretics, lithium, estrogens, fluorides, glucose, and insulin. Inaddition, serum calcium fluctuations are a side effect of many existingbone-related therapies, such as bisphosphonate and parathyroid hormonetherapy. Because of the potentially life-threatening consequences ofcalcium imbalance, patients susceptible to hypocalcemia or hypercalcemiamay need to forego certain therapy options.

Accordingly, the materials and method of the invention, particularlycombinations, are advantageous in treating patients that are susceptibleor sensitive to unstable calcium levels. The amount of sclerostinbinding agent administered to a human in the context of this aspect ofthe invention is an amount that does not result in hypocalcemia orhypercalcemia (e.g., clinically-significant hypocalcemia orhypercalcemia). In addition, the invention provides a method of treatinga bone-related disorder in a human suffering from or at risk ofhypocalcemia or hypercalcemia or a human in which treatment withbisphosphonate, a parathyroid hormone, or parathyroid hormone analog iscontraindicated. The method comprises administering to the human anamount of a sclerostin binding agent effective to increase the level ofa marker of bone formation, such as serum levels of BSAP, P1NP, and/orOstCa and/or reduce the level of a marker of bone resorption, such asCTX.

Further provided herein are methods of treating or preventing loss ofbone mass comprising administering to a patient in need thereof atherapeutically effective amount of an antibody comprising a variableregion selected from SEQ ID NOs: 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64,66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94 and 96, orimmunologically functional fragment thereof as described herein (e.g.,an antibody or immunologically functional fragment that comprises atleast one light chain CDR selected from the group consisting of SEQ IDNOs: 97 to 227 and 228. In one aspect of this embodiment, the patient isone who suffers from cancer that metastasizes to bone, and in anotheraspect, the patient is one who suffers from multiple myeloma. One ofskill in the art will appreciate that these compositions, alone or incombination with one or more other therapeutics, can be useful for theformulation of a medicament. Antibodies of the present invention aresuitable for treatment of bone related disorders. The antibody depictedin SEQ ID NOs: 42 and 44 for use in treatment of bone fracture. Theantibody depicted in SEQ ID NOs: 42 and 44 for use in treatment of gapunion defects. The antibody depicted in SEQ ID NOs: 42 and 44 incombination with an inhibitory sclerostin antibody for use in treatmentof bone fracture. The antibody depicted in SEQ ID NOs: 42 and 44 incombination with an inhibitory sclerostin antibody for use in treatmentof gap union defects. It is understood that the term ‘bone fracture’ ismeant to include one or more fractures in a patient in need oftreatment.

Particular conditions which may be treated by the compositions of thepresent invention include dysplasias, wherein growth or development ofbone is abnormal. Representative examples of such conditions includeachondroplasia, cleidocranial dysostosis, enchondromatosis, fibrousdysplasia, Gaucher's Disease, Marfan's syndrome, multiple hereditaryexotoses, neurofibromatosis, osteogenesis imperfecta, osteopetrosis,osteopoikilosis, sclerotic lesions, pseudoarthrosis, and pyogenicosteomyelitis.

Other conditions that may be treated or prevented include a wide varietyof causes of osteopenia, osteoporosis and bone loss. Representativeexamples of such conditions include periodontal disease, anti-epilepticdrug induced bone loss, primary and secondary hyperparathyroidism,familial hyperparathyroidism syndromes, weightlessness induced boneloss, osteoporosis in men, postmenopausal bone loss, osteoarthritis,renal osteodystrophy, infiltrative disorders of bone, oral bone loss,osteonecrosis of the jaw, juvenile Paget's disease, melorheostosis,metabolic bone diseases, mastocytosis, sickle cell disease, ischemicbone disease (such as Legg-Calve-Perthes disease, regional migratoryosteoporosis), anemic states, conditions caused by steroids,glucocorticoid-induced bone loss, heparin-induced bone loss, bone marrowdisorders, scurvy, malnutrition, calcium deficiency, idiopathicosteopenia or osteoporosis, congenital osteopenia or osteoporosis,alcoholism, chronic liver disease, postmenopausal state, chronicinflammatory conditions, rheumatoid arthritis, inflammatory boweldisease, ulcerative colitis, inflammatory colitis, Crohn's disease,oligomenorrhea, amenorrhea, pregnancy, diabetes mellitus,hyperthyroidism, thyroid disorders, parathyroid disorders, Cushing'sdisease, acromegaly, hypogonadism, immobilization or disuse, reflexsympathetic dystrophy syndrome, regional osteoporosis, osteomalacia,bone loss associated with joint replacement, HIV associated bone loss,bone loss associated with loss of growth hormone, bone loss associatedwith cystic fibrosis, fibrous dysplasia, chemotherapy associated boneloss, tumor induced bone loss, cancer-related bone loss, hormoneablative bone loss, multiple myeloma, drug-induced bone loss, anorexianervosa, disease associated facial bone loss, disease associated cranialbone loss, disease associated bone loss of the jaw, disease associatedbone loss of the skull, and bone loss associated with space travel.Further conditions relate to bone loss associated with aging, includingfacial bone loss associated with aging, cranial bone loss associatedwith aging, jaw bone loss associated with aging, and skull bone lossassociated with aging.

As permitted in certain national jurisdictions, references disclosedherein are hereby incorporated by reference in their entireties as ifeach was incorporated individually, for any purpose including enablingand describing the invention. The following examples are provided solelyto illustrate certain aspects of the antibodies, fragments andcompositions that are provided herein and thus should not be construedto limit the scope of the claimed invention.

EXAMPLES Example 1

Preparation of the Human DKK1 (huDKK1) Immunogen

The cloning of human DKK1 was as described in U.S. Pat. No. 6,344,541with the following modifications. Two different epitope tagged versionsof human DKK1 were used as an immunogen, one contained the FLAG epitopeand the other was an fc-fusion molecule. Both epitope tags were appendedto the carboxy-terminus of human DKK1 using standard molecular biologytechniques obvious to those skilled in the art.

The epitope tagged versions of human DKK1 were cloned into an expressionvector for expression in CHO cells. Human DKK1 variants containingeither the FLAG or Fc epitopes were purified from conditioned medium foruse as an antigen to generate anti-huDKK1 antibodies. Epitope-taggedhuDKK1 was purified from concentrated conditioned media (CM). Otherprotein production and purification procedures known to those skilled inthe art may also be used.

Example 2 Immunization and Titering

Recombinant FLAG-tagged human DKK1 (FLAG-DKK1) and recombinant Fc-taggedhuman DKK1 (DKK1-fc) were used as antigens. Monoclonal antibodiesagainst DKK1 were developed by sequentially immunizing XenoMouse® mice(Abgenix, Inc. Fremont, Calif.) (see, e.g., U.S. Pat. No. 7,435,871 andthe description therein). XenoMouse animals were immunized via footpadroute for all injections. Anti-DKK antibody titers in the serum fromimmunized XenoMouse mice were determined by ELISA.

Example 3 Recovery of Lymphocytes, B-Cell Isolations, Fusions andGeneration of Hybridomas

Lymph nodes were harvested and pooled from each cohort. The totaleffluent was collected as the CD90-negative fraction (most of thesecells were expected to be B cells). The fusion was performed by mixingwashed enriched B cells from above and nonsecretory myeloma P3X63Ag8.653cells purchased from ATCC, catalogue CRL 1580 (Kearney et al, J.Immunol. 123, 1979, 1548-1550) at a ratio of 1:1. Electro-cell fusion(ECF) was performed using a fusion generator, model ECM2001, Genetronic,Inc., San Diego, Calif. The fusion chamber size used was 2.0 mL

After ECF, the cell suspensions were carefully removed from the fusionchamber under sterile conditions and transferred into a sterile tubecontaining the same volume of Hybridoma Culture Medium (DMEM (JRHBiosciences)). The cells were incubated and then centrifuged. The cellswere resuspended in a small volume of Hybridoma Selection Medium(Hybridoma Culture Medium supplemented with 0.5× HA (Sigma, catalogueA9666)), and the volume was adjusted appropriately with more HybridomaSelection Medium. The cells were mixed gently and pipetted into 96-wellplates and allowed to grow.

Example 4

After sufficient culture, hybridoma supernatants were screened forDKK1-specific monoclonal antibodies. In the Primary screen, the ELISAplates were coated with 50 μL/well of Flag tagged rhDKK1 (2 μg/mL) thenincubated at 4° C. overnight. After incubation, the plates were washedwith Washing Buffer three times and then 200 μL/well Blocking Bufferwere added and the plates were incubated at room temperature. Afterincubation, the plates were washed with Washing Buffer three times.Aliquots (50 μL/well) of hybridoma supernatants and positive andnegative controls were added, and the plates incubated at roomtemperature for 2 h.

After incubation, the plates were washed with washing buffer. FiftyμL/well of detection antibody was added and the plates incubated at roomtemperature for 1 hour. After incubation, the plates were washed threetimes with Washing Buffer and then 50 μL/well of TMB was added, and theplates were allowed to develop for approximately 10 minutes (untilnegative control wells barely started to show color). 50 μL/well stopsolution was then added and the plates were read on an ELISA platereader at a wavelength of 650 nm. The cutoff point OD was set attwo-fold above the OD of the negative control.

The old culture supernatants from the positive hybridoma cells growthwells based on primary screen were removed completely and the DKK1positive hybridoma cells were suspended with fresh hybridoma culturemedium and transferred to 24-well plates. After 2 days a secondaryconfirmation screen was conducted where the positive hybridomas in thefirst screening were confirmed in Flag tagged rhDKK1 coated ELISA(described as above) and Flag tagged irrelevant antigen coated ELISA.Three sets of detection system for antigen coated ELISA, one set forhIgG detection, one set for human Ig kappa light chain detection and theother set for human lambda light chain detection in order to demonstratefully human composition for both IgG and Ig kappa or IgG and Ig lambdaor IgG and Ig kappa plus lambda. Only hIgG detection was used forirrelevant antigen coated ELISA. The three sets of antigen coated ELISAprocedures were identical to the descriptions above except the threedifferent detection antibodies were used separately. Final selection wasbased on a positive signal on antigen and a negative signal onirrelevant antigen.

The human IgG/kappa or IgG/lambda DKK1 specific monoclonal antibodiesgenerated are detailed in Table 2.

TABLE 2 Human anti- XenoMouse Strain DKK1 Cohort (Human Isotype)Immunogen IgG Abs IgGκ IgGλ 1 XMG4 (IgG4) FLAG-DKK1 5 5 N.A. 2 XMG2(IgG2) FLAG-DKK1 42 42 N.A. 3 XMG2-KL (IgG2) FLAG-DKK1 7 0 7 4 XMG2-KL(IgG2) DKK1-fc 6 1 5 5 XMG4-KL (IgG4) FLAG-DKK1 85 43 42 6 XMG2-KL(IgG2) FLAG-DKK1 158 91 68 7 XmG4-KL (IgG4) FLAG-DKK1 41 19 23

Hybridomas secreting antibodies deemed to be of functional interest weresingle-cell cloned by limiting dilution. Screening of single-cell clonedhybridomas for DKK1 antibody was performed by ELISA as described above.Hybridoma clones were cultured in Hybridoma Culture Medium and expandedusing standard tissue culture techniques to produce exhausted culturesupernatant containing secreted monoclonal antibody. Freezer stocks ofhybridoma clones were also generated.

Example 5 Selection of Hybridomas Producing Neutralizing Antibodies toHuman DKK1 by Bioactivity

The hybridomas obtained as described in Example 2 were tested utilizinga TCF/lef-luciferase reporter construct in which luciferase expressionis under the control of the canonical Wnt pathway. When cellstransfected with this construct are exposed to biologically active Wnt,luciferase activity is induced. The Wnt induced luciferase activity canbe suppressed by adding recombinant DKK1 protein to the cells thatcontain this construct. For the present experiments, both Wnt3a and DKK1first were added to the cells in amounts optimized to suppress about 80%of the Wnt-dependent luciferase expression. The further addition of ananti-DKK1 antibody with neutralizing activity to these same cells isexpected to restore Wnt activity, thus resulting in increased luciferaseexpression. Supernatants from the hybridomas were thus tested todetermine whether they were capable of restoring luciferase expressionin cells transfected with the Wnt/luciferase construct. Luciferaseactivity was quantified as described below.

On day zero, freshly trypsinized 293T cells were plated onfibronectin-coated 96 well plates. The cells were then co-transfectedwith DNA encoding firefly luciferase and DNA encoding renillaluciferase. On day 1, for each well, TCF/lef-luciferase DNA and 1 ngrenilla luciferase DNA in 30 μl of DMEM were mixed with PolyfectTransfection Reagent™ (Qiagen 301107) and incubated for 10 minutes atroom temperature to allow formation of a PolyFect-DNA complex. Followingthis incubation, 100 μl of growth medium were added to the complex. Thenthe culture medium was removed from each well and the complex in growthmedium was added to the well. The growth medium in the wells was removedthree hours later and replaced with conditioned medium containing Wnt3a,recombinant human DKK1 and anti-DKK1 hybridoma conditioned medium.

After three days, the cells were washed once with PBS, and to each wellwere added passive lysis buffer. Plates were shaken for 20 minutes atroom temperature to induce lysis. Ten μl of lysate per assay were usedto perform the assay in 96 well white plates according to themanufacturer's protocol. Luminescent signals from firefly and renillaluciferases were both recorded and the ratio of those signals was usedto determine the EC50 and to plot dose-response curves. First, thesubstrate of firefly luciferase was injected into a well with celllysate and the luminescent signal recorded; then the substrate ofrenilla luciferase was injected into the same well and the resultingsecond luminescent signal was recorded.

The stromal cell line ST2, derived from mouse bone marrow, was used asan additional screen for isolating anti-DKK1 antibodies withneutralizing activity. In response to Wnt3a signaling, ST2 cellsdifferentiate into osteoblasts which express the osteoblast markerprotein alkaline phosphatase (ALP). The induction of ALP by Wnt3a inthese cells can be blocked by adding the Wnt inhibitor DKK1 to theculture medium. ALP expression can be restored under these conditions byexposing the cells to an agent capable of neutralizing DKK1 activity,such as a neutralizing anti-DKK1 antibody.

In summary, of the hybridomas were screened in the ELISA assay, 344bound DKK1 in the ELISA assays and 25 were positive in one or both ofthe neutralization assays (TCR/lef reporter assay or ST2 cell assay).The hybridomas exhibiting the best activity from each of the threecampaigns are shown in table 3. As can be seen from the cellularactivities of these antibodies those derived from the second (5.X.x) andthird (6.X.x) campaigns, that is those generated in the KL mice, ingeneral exhibited better cell-based activities as is evident from thelower EC50s than those generated in the first (2.X.x) campaign with theXenomouse.

TABLE 3 Antibody TCF EC50 (nM) ST2 EC50 (nM) 2.20.1 10.67 34.67 2.37.140.18 145.33 2.4.1 38.59 17.2 2.40.1 1.28 2.47 2.41.1 32.07 89.07 2.47.160.7 NA 5.17.1 3.48 8.24 5.23.1 1.01 4.29 5.25.1 1.88 5.96 5.31.1 5.2910.38 5.32.1 1.01 3.48 5.40.1 3.75 7.24 5.65.1 4.21 5.87 5.76.1 2.554.09 5.77.1 2.14 6.23 5.78.1 3.75 5.70 5.80.1 3.22 5.03 5.85.1 4.5310.55 6.116.6 4.69 2.77 6.139.5 9.78 3.93 6.147.4 3.95 2.57 6.37.5 6.573.98

Example 6 Cloning and Sequence Analysis of Antibodies

Total RNA was prepared from anti-DKK1 hybridoma cell lines. DNAsequences were provided by Abgenix or obtained by sequencing of clonedRACE (rapid amplification of cDNA ends) PCR (polymerase chain reaction)products.

Example 7 Expression and Purification of Human Anti-huDKK1 Antibodies inCHO Cells

The anti DKK1 cell lines were created by transfecting CHO host cellswith the expression plasmids pDC323-anti DKK1 kappa and pDC324[anti-DKK1-IgG2] for 2.40.3, 6.35.5, 6.116.6 HC-IgG2 and LC-kappa usinga standard electroporation procedure. After transfection of the hostcell line with the expression plasmids the cells were grown in—GHTselection medium containing 4% dialysed fetal bovine serum (ds or dfFBS)for 2-3 weeks to allow for selection of the plasmid and recovery of thecells. Serum was then removed from the medium and the cells were grownin GHT selective medium until they achieved >85% viability. This pool oftransfected cells was then cultured in medium containing [150-300] nMMTX followed by medium containing 500-1000 nM MTX to select for highexpressing cells.

Anti-huDKK1-1 antibody expressing cell lines were expanded using asepticcell culture techniques. Cells were inoculated into a bioreactor uponexpansion and the culture fed as needed. At harvest, cells werecentrifuged and conditioned media filtered. Human anti-huDKK1 antibodieswere purified from conditioned medium on Protein A sepharose. PurifiedDKK1 antibody was buffer exchanged to the buffer of choice.

Example 8 ELISA-Based Cross Blocking Assay

Liquid volumes used in this example were those typically used in 96-wellplate ELISAs (e.g. 50-200 ul/well). Ab-X and Ab-Y, in this example wereassumed to have molecular weights of about 145 Kd and to have 2 DKK1binding sites per antibody molecule. An anti-DKK1 antibody (Ab-X) wascoated (e.g. 50 ul of 1 ug/ml) onto a 96-well ELISA plate for at leastone hour. After this coating step the antibody solution was removed, theplate washed with wash solution and was then blocked using anappropriate blocking solution and procedures known in the art. Blockingsolution was removed from the ELISA plate and a second anti-DKK1antibody (Ab-Y), which was being tested for its ability to cross-blockthe coated antibody, was added in excess (e.g. 50 ul of 10 ug/ml) inblocking solution to the appropriate wells of the ELISA plate.

Following this, a limited amount (e.g. 50 ul of 10 ng/ml) of huDKK1 inblocking solution was then added to the appropriate wells and the platewas incubated for at least one hour at room temperature while shakingand the plate was then washed. An appropriate amount of a DKK1 detectionreagent in blocking solution was added to the ELISA plate and incubatedfor at least one hour at room temperature.

The plate was then washed with wash solution and developed with anappropriate reagent. The background signal for the assay was defined asthe signal obtained in wells with the coated antibody (in this caseAb-X), second solution phase antibody (in this case Ab-Y), DKK1 bufferonly (i.e. no DKK1) and DKK1 detection reagents. The positive controlsignal for the assay was defined as the signal obtained in wells withthe coated antibody (in this case Ab-X), second solution phase antibodybuffer only (i.e. no second solution phase antibody), DKK1 and DKK1detection reagents. The ELISA assay needs to be run in such a manner soas to have the positive control signal be at least 6 times thebackground signal.

Ab-X and Ab-Y are defined as cross-blocking if, either in format 1 or informat 2, the solution phase anti-DKK1 antibody was able to cause areduction of between 60% and 100%, specifically between 70% and 100%,and more specifically between 80% and 100%, of the DKK1 detection signal(i.e. the amount of DKK1 bound by the coated antibody) as compared tothe DKK1 detection signal obtained in the absence of the solution phaseanti-DKK1 antibody (i.e. the positive control wells). It will beunderstood by one of skill in the art that the term ‘cross block’ is notintended to encompass only complete blockage of binding of the testmolecule, rather it can include a range of decreased binding less than100% as described here. In one example, an isolated antibody or fragmentthereof that cross blocks the binding of antibody depicted in SEQ IDNOs: 42 and 44 to human DKK1 and/or is cross-blocked from binding tohuman DKK1 by the antibody depicted in SEQ ID NOs: 42 and 44. Antibodiesthat are cross blocked from binding human DKK1 by the binding ofantibody depicted in SEQ ID NOs: 42 and 44 include those that have 60%reduction in binding to human DKK1, 70% reduction in binding to humanDKK1, 80% reduction in binding to human DKK1, 90% reduction in bindingto human DKK1 or 95% reduction in binding to human DKK1. Antibodies thatcross block binding of human DKK1 by the binding of antibody depicted inSEQ ID NOs: 42 and 44 include those that reduce its binding by 60% inbinding to human DKK1, 70% reduction in binding to human DKK1, 80%reduction in binding to human DKK1, 90% reduction in binding to humanDKK1 or 95% reduction in binding to human DKK1. Antibodies that werecapable of cross blocking one another are referred to herein as being inthe same bin.

In the event that a tagged version of DKK1 is used in the ELISA, such asa N-terminal His-tagged DKK1 (R & D Systems, Minneapolis, Minn., USA;2005 catalog number 1406-ST-025) then an appropriate type of DKK1detection reagent would include an HRP labeled anti-His antibody. Inaddition to using N-terminal His-tagged DKK1, one could also useC-terminal His-tagged DKK1. Furthermore, various other tags and tagbinding protein combinations that are known in the art could be used inthis ELISA-based cross-blocking assay (e.g. HA tag with anti-HAantibodies; FLAG tag with anti-FLAG antibodies; biotin tag withstreptavidin).

The human anti-huDKK1 neutralizing antibodies described herein recognizetwo distinct epitopes as evident by the inability of the antibodies tocross block one another. The first epitope is referred to as 11H10 thathas been described previously (U.S. Pat. No. 7,709,611). The secondepitope is described below and is referred to as 5.25.1 (SEQ ID NOs: 42and 44).

Example 9

Characterization of Human DKK1 Epitopes that Bind 5.25.1 Antibody

Human DKK1 contains two disulfide-rich domains located near theN-terminus and at the end of the C-terminus, referred to here as the N-and C-terminal disulfide domains. The N-terminal disulfide domain(hereinafter, “disulfide domain 1” or “D1”) contains 55 amino acidsresidues (amino acids 85-139 of SEQ ID NO:2) and has 10 cysteinesforming 5 intramolecular disulfide bonds. The C-terminal disulfidedomain (hereinafter, “disulfide domain 2”, or “D2”) contains 75 aminoacids (amino acids 189-263 of SEQ ID NO:2) and contains 10 cysteinesthat also form 5 intramolecular disulfide bridges. These two disulfidedomains are separated by a stretch of about 50 amino acids. Disulfidedomain 2 (D2) of DKK1 has been proposed to have a molecular structuresimilar to the canonical colipase fold, the crystal structure of whichhas been determined using porcine colipase (Aravind, A. and Koonin, E.V., Current Biology 8:R477-479 (1998)). Intramolecular disulfidelinkages among the 10 cysteine residues in the N-terminal D1 domain ofDKK molecule have recently been determined.

Treatment with a reducing agent abolished the ability of DKK1 to bind5.25.1, thus indicating that the epitope targeted by this antibody isconformational (or discontinous) and requires the maintenance of intactdisulfide bonds in the D1 and D2 domains. To characterize thisconformational epitope, a strategy was applied that involved fragmentinghuman DKK1 with cyanogen bromide (CNBr) and several different proteases,then analyzing the fragments and testing them for their ability to bindto the antibody. Digestion was also performed in the presence of 5.25.1to detect those amino acid residues or sequence regions that isprotected from proteolysis due to antibody binding. The resulting datapermitted the location of the epitope(s) to be elucidated. In essence,the DKK1 proteolytic digestion was carried out in the absence orpresence of antibody 5.25.1 and then subjected to HPLC peptide mapping.A partial or complete reduction in the height of an HPLC peak and/or thedetection of a newly generated peak in a sample exposed to antibody maybe observed.

After each peptide digest, the reaction products were separated by HPLC,the individual peaks were collected and the peptides identified andmapped by N-terminal sequencing. To determine if the peptides could bind5.25.1, they were subjected to real time biospecific interaction assayswith a BiaCore workstation, using a sensorgram surface covalently boundwith HuDKK1 as a biosensor for binding. HPLC peptide mapping wasperformed under standard conditions.

CNBr Digestion

CNBr cleavage of hDKK1 generated two large fragments. These were CNBr1and CNBr2, which represented, respectively, D2 and D1 disulfide domains.CNBr1 consisted of two peptides (amino acids 179-206 of SEQ ID NO:2 andamino acids 207-266 [or 274 if including the added C-terminal flagpeptide] of SEQ ID NO:2) held together by 5 disulfide bonds. CNBr2similarly consisted of two peptides (amino acids 32-122 of SEQ ID NO:2and amino acids 127-178 of SEQ ID NO:2), also held together by 5disulfide bonds (Table 4). The results of BiaCore analysis indicatedthat 5.25.1 was capable of binding significantly to CNBr2 but did notbind at all to CNBr1. Thus, it was concluded that 5.25.1 binds to anepitope region located in D1 disulfide domain of HuDKK1.

Trypsin Digestion

Human DKK1 was next digested with trypsin, which cleaves after ARG andLYS residues. About 200 μg of DKK1 at 0.5-1.0 mg/ml were incubated inPBS (pH 7.2) for 20 h at 37° C. with 8 μg of one or the other of theseproteases to achieve complete digestion of the DKK1.

HPLC chromatography of tryptic digests yielded multiple peaks, whichwere collected, dried, and reconstituted into 0.1M sodium phosphatebuffer, pH 7.2. Table 4 depicts DKK1 Peptide fragments containing theN-terminal disulfide domain D1 derived from CNBr cleavage andproteolytic digestions.

TABLE 4 Sequence positions with Fragment Number of peptides reference toSEQ ID NO: 2 CNBr2 2 32-122 and 127-178 T2 (or T3)¹ 5 74-102, 103-115,121-123, 124-134, & 135-147 T4² 1 74-147 AspN1 (AspN2) 2 78-104 &105-141 ¹Peptides were derived from trypsin digestion of HuDKK1 alone.²Peptide is derived from trypsin digestion of HuDKK1 in the presence of5.25.1 antibody.

Sequence analysis was conducted on the peptide peaks recovered from HPLCafter trypsin digestion. Peptide peaks containing peptide sequenceswithout disulfide linkages were also confirmed by LC-MS/MS analysis.Molecular mass of fragments containing multiple disulfide-linkedpeptides was confirmed by matrix-assisted laser desorption massspectrometric analysis (MALDI-MS). Two peaks, T2 (retention time 40.7min using 1 mm i.d. column or 43.5 minutes using 2.1 mm column) and T3(retention time 41.9 min using 1 mm i.d. column or 44.7 minutes using2.1 mm column), were confirmed to contain sequences that mapped todisulfide domain 1, while T1 peptide (retention time 35 min in 1 mm C18column or 36.5 min in 2.1 C4 column) mapped to disulfide domain D2. Noneof T1, T2, and T3 bound to 5.25.1 when tested by BiaCore bindingexperiments. T2 and T3 are large peptide fragments consisted of fivesmall peptides (3 to 13 amino acids in length) held together by the fivedisulfide bonds in D1 domain with amino acids 74-102, 103-115, 121-123,124-134, 135-147 of SEQ ID:2 (Table 4). One small segment of thesequence at disulfide domain 1 was missing from T2 and T3. This missingsequence containing all Lys and Arg, was amino acids 116-120 (sequenceof ARG-ARG-LYS-ARG) of SEQ ID NO:2.

Human DKK1 was also incubated with 5.25.1 at a molar ratio of 1:1 to 1:3in PBS buffer for 1 h at room temperature. Aliquot of the DKK1/antibodycomplex was then digested by trypsin under conditions as describedabove. HPLC peptide mapping profile of the tryptic digest is completelyidentical to that obtained from the DKK1 digest in the absence ofantibody 5.25.1, except that T2 and T3 peaks disappeared and a new peakT4 (retention time 41.3 minutes using 1 mm column or 44.3 minutes using2.1 mm i.d. column) became detectable. T4 is also a the N-terminaldomain D1 peptide, but only contains a single amino acid sequence withamino acids 74-147 of sequence ID NO:2 (Table 4). In Biacore bindingassay, T4 binds to 5.25.1 and can compete for 5.25.1 binding withSensorchip surface-bound DKK1.

AspN Digestion

To further delineate the 5.25.1-binding epitope, HuDKK1 was digestedwith protease AspN and the resulting fragments analyzed as describedabove. Of the major HPLC peaks generated by AspN digestion, peaks thatbound antibody 5.25.1 were AspN1, AspN2 and AspN3. Sequence analysisindicated that AspN1 and AspN2 were derived from disulfide domain D1.AspN1 and AspN2 were identical in amino acid sequence and each of themconsisted of two peptides held together by five disulfide bonds indisulfide domain D1. These two peptides consisted of amino acids 78-104and 105-141 of SEQ ID NO:2 (see Table 4). AspN3 is a partial digestionproduct whose sequence contains both Domains D1 and D2 sequences. Twoother peaks, AspN4 and AspN5, were also isolated and confirmed to bedisulfide-linked peptides in domain D2, AspN4 or AspN5 does not competewith DKK1 for 5.25.1 binding.

Analysis of Digestion Results

The above results indicate that 5.25.1 binds to non-linear epitopes ofhuman DKK1 located in disulfide domain D1. As exemplified in FIG. 1,epitope regions are deduced with the observations described in thefollowing:

Trypsin cleavage (R at position 102, and RKRRKR between positions 115and 120 and K at position 134 in FIG. 1) generates five peptides linkedby disulfides. This D1 domain tryptic peptide fraction loses 5.25.1binding activity.

Antibody 5.25.1 can bind to DKK1 to protect all cleavage sites on D1domain from trypsin proteolysis (R at position 99, and RKRRKR betweenpositions 115 and 120 and K at position 134). The obtained D1 trypticfragment, recovered at a distinct retention time, is a singlepolypeptide chain, indicating that all Arg and Lys's in D1 are protectedfrom proteolysis and therefore are closely located at the epitope regionor involved in epitope binding. Binding activity is maintained in D1fragments generated Asp N or CNBr cleavage. To maintain bindingactivity, the observed minimal fragment size for D1 domain is aminoacids 78-141, except that Asp-N had clipped the peptide bond between Gly105 and Asp at position 105, leaving this large disulfide loop (formedbetween Cys 97 and Cys 111) not being connected together.

CNBr cleavage for removal of amino acids 123-126 (sequence ofARG-HIS-ALA-MET) and AspN cleavage of Gly114-Asp115 peptide bond do notinfluence CNBr2 and AspN1 (or AspN2) fragments from 5.25.1 binding,therefore, sequences in these region are not in the epitope. A highlynegatively charged region (amino acids 83-91) in D1 is resistant to GluCand AspN digestion in the absence of Ab, indicating that this region isnot accessible for proteolysis (may be due to steric hindrance) and isalso inaccessible to 5.25.1.

In summary, epitopes residing in HuDKK1 for 5.25.1 binding includediscontinuous sequences at the N-terminal disulfide domain D1: aminoacids 98-104, 107-121, and 129-140 of SEQ ID NO:2. And the D1 domaindisulfide bonds have to remain intact to retain a correct conformationor three-dimensional structure for 5.25.1 to bind.

Example 10 Binding Affinity of Monoclonal Antibodies Against DKK1

Analyses were performed to study the binding of human anti-huDKK1antibodies to DKK1 using BiaCore 2000 (BIACORE, Uppsala, Sweden).BiaCore allowed us to determine the kd of the selected antibodies. Thoseantibodies with a lower kd are more desirable as they bind hDKK1 longerthan those with a larger kd, and thus are more likely to engender agreater response. The binding sensorgrams were analyzed and the data aresummarized below.

TABLE 5 Antibody kd (1/s) 2.20.1 2.40E−04 2.37.1 2.40E−03 2.4.1 6.00E−042.40.1 6.27E−05 2.41.1 1.30E−03 2.47.1 9.20E−04 5.17.1 3.03E−04 5.23.11.81E−04 5.25.1 <2e−5 5.31.1 1.05E−04 5.32.1 1.91E−04 5.40.1 2.81E−045.65.1 5.25E−04 5.76.1 2.41E−04 5.77.1 <2e−5 5.78.1 1.91E−04 5.80.1<2e−5 6.116.6 2.00E−05 6.139.5 2.09E−04 6.147.4 2.51E−04 6.37.5 4.00E−05

In addition to the off rate, other parameters such as ka (on-rate), KD(affinity), cell-based and in vivo activity are also factors influencingoverall selection of therapeutics. The data in table 5 also indicatedthat those antibodies derived from the latter immunizations of the KLmice yielded antibodies with more desirable Kd's. The binding to huDkk4was also tested for several of the antibodies to determine specificityand it was determined that human anti-huDKK1 antibodies had at least a50-fold increased specificity towards DKK1 than to Dkk4, with 5.25.1 and5.32.1 exhibiting no detectable binding to Dkk4.

Interestingly when the sensograms generated from the BiaCore analysis ofthe second campaign antibodies, which contained antibodies in both the11H10 and 5.25.1 bins, were analyzed it became apparent that differencesexist between the bins. The antibodies from the 11H10 bin, at a givenantibody concentration, gave a higher binding signal than did antibodiesfrom the 5.25.1 bin. An increased maximal signal is observed from the11H10 bin antibodies (2.40.2 and 5.80.2 and 5.80.3).

It was apparent from the BiaCore results that the human anti-huDKK1antibodies varied in affinity for DKK1, and that the affinity forseveral of these to human DKK1 exceeded the sensitivity limits of theBiaCore assay. Accordingly, the affinity of several of these antibodiesto DKK1 was further assessed by an equilibrium binding analysis usingthe more sensitive KinExAtm 3000. For these measurements, Reacti-Gel 6×beads (Pierce, Rockford, Ill.) were pre-coated with human, cynomologousor mouse DKK1 and blocked with BSA. One hundred pM, 300 pM, or 1000 pMof the antibody was mixed with various concentrations of human, mouse orcyno DKK1, ranging in concentration from 1 pM to 50 nM, and equilibratedat room temperature for 8 hours. The mixtures were then passed over theDKK1-coated beads. The amount of bead-bound anti-DKK1 antibody wasquantified using goat anti-human-IgG antibody labeled with a fluorescenttag (Cy5; Jackson Immuno Research, West Grove, Pa.). The amount offluorescent signal measured was proportional to the concentration offree anti-DKK1 antibody in each reaction mixture at equilibrium. Thedissociation equilibrium constant (Kd) was obtained from nonlinearregression of the competition curves using a dual-curve one-sitehomogeneous binding model using the KinExA software. Results of theKinExA assays for the selected antibodies are shown in table 6.

TABLE 6 Antibody K_(D human) (pM) K_(D mouse) (pM) K_(D cyno) (pM)2.40.1 220 480 220 5.25.1 3 150 5.32.1 3 40 5.77.1 8 140 5.80.1 60 256.116.6 25 40 40 6.139.5 110 6.147.4 125 6.37.5 30 50 35

Example 11

Only 11H10 Bin Antibodies Block Binding of huDKK1 to LRP6 and Kremin2

The ability of 11H10 bin and 5.25.1 bin antibodies to block binding ofDKK1 to the Wnt co-receptor LRP6 or to Kremin2 was examined usingco-immunoprecipitation procedures. Recombinant mouse LRP6-His andrhDKK1-Flag or recombinant human kremen2-his and hDKK1-flag werepre-incubated with or without the anti-DKK1 antibody in HANKs balancedsalt solution with shaking overnight to allow complex formation.

In FIG. 2A rhDKK1-flag was incubated with LRP6-His and 5 μg of one ofthe neutralizing DKK1 antibodies from either the 11H10 bin (5.80.1,6.37.5 or r11H10) or the 5.25.1 bin (5.25.1, 5.77.1). The mixture wasimmunoprecipitated with an anti-his antibody that would bind his-taggedLRP6 and pull down associated DKK1. The immunoprecipitated was thensubjected to Western blotting analysis using an anti-flag antibody thatrecognized rhDKK1. In this way DKK1 associated with LRP6 in solution andthe ability of neutralizing-DKK1 antibodies to compete for the bindingof DKK1 to LRP6, and by inference to LRP5, could be measured. In lane 1only LRP6-His is included; lane 2 rhDKK1-Flag; lane 3hLRP6-His+hDKK1-Flag; lane 4 hLRP6-His+hDKK1-Flag+5.80.1; lane 5hLRP6-His+hDKK1-Flag+6.37.5; lane 6 hLRP6-His+hDKK1-Flag+r11H10; lane 7hLRP6-His+hDKK1-Flag+5.25.1; lane 8 hLRP6-His+hDKK1-Flag+5.77.1. Thedata indicated that all three 11H10 bin antibodies, but not the 5.25.1bin antibodies, can block the interaction of DKK1 to LRP6.

In a similar manner the ability of the same aforementioned antibodies toblock the binding of DKK1 to Kremin2, and by inference to Kremin1, wasdetermined (FIG. 2B). In lane 1 only LRP6-His is included; lane 2rhDKK1-Flag; lane 3 hLRP6-His+hDKK1-Flag; lane 4hLRP6-His+hDKK1-Flag+0.5 μg 5.80.1; lane 5 hLRP6-His+hDKK1-Flag+5 μg5.80.1; lane 6 hLRP6-His+hDKK1-Flag+0.5 μg 6.37.5; lane 7hLRP6-His+hDKK1-Flag+5 μg 6.37.5; lane 8 hLRP6-His+hDKK1-Flag+0.5 μgr11H10; lane 9 hLRP6-His+hDKK1-Flag+5 μg r11H10; lane 10hLRP6-His+hDKK1-Flag+5 μg 5.25.1; lane 11 hLRP6-His+hDKK1-Flag+5 μg5.77.1.

The data indicated that all three 11H10 bin antibodies, but not the5.25.1 bin antibodies, can block the interaction of DKK1 to Kremin2. Thedata presented in this experiment suggest that the two differentantibody bins exhibit different mechanism of actions in the ability toneutralize DKK1 activity on Wnt signaling.

Example 12 In Vivo Activity of Selected Antibodies

Experiments were conducted to determine whether neutralization of DKK1in a mouse animal model would cause an increase in bone mineral density(BMD) and in serum osteocalcin, a marker for bone formation. Theantibodies tested were 2.40.2, 5.32.5, 5.80, 6.37.5, 6.116.6 and werepurified as described above.

In the first experiment four-week-old male BDF-1 mice (APR 233757,Charles River) were injected subcutaneously over a three-week periodwith one of three doses of the purified 2.40.2 monoclonal antibody (5,10, or 20 mg/kg). Five mice were used per group. Negative control micewere injected with vehicle (PBS), and positive control mice wereinjected with parathyroid hormone (amino acids 1-34), which is known tostimulate increased bone density in these mice (Dempster et al.,Endocrine Reviews 14(6):690-709 (1993)). One hundred μg/kg of PTH (1-34)was used per injection. The results for percent change in tibia bonemineral density at three weeks are shown below in FIG. 3.

To compare the in vivo efficacy of the two different antibody bins,representative antibodies were selected from each bin. The antibodiesselected were 5.32.1 from the 5.25.1 bin and 5.80.1 from the 11H10 bin.Both these antibodies bound mouse DKK1 with similar affinities.Eight-week-old male BDF-1 mice (APR 233757, Charles River) were injectedsubcutaneously over a two-week period with one of three doses of thepurified monoclonal antibody (3, 10, or 30 mg/kg). Six mice were usedper group. Negative control mice were injected with vehicle (PBS). Thedata are presented in FIG. 4 as percent change from baseline in bonemineral density of the lumbar vertebrae and indicate that the 11H10 binantibody (5.80.1) exhibits superior bone building activity than the5.25.1 bin antibody (5.32.1).

In another experiment two additional antibodies from the 11H10 bin wereinjected into 8-week old, male BDF-1 mice. These mice were injectedsubcutaneously twice per week for three weeks with 25 mg/kg of therespective antibodies (6.37.5 and 6.116.6). Ten mice were used pergroup. Control groups were injected with vehicle (twice per week) or PTH(100 μg/kg five times per week). The data are presented below in FIG. 5as percent change from baseline in bone mineral density of the lumbarvertebrae and indicated that these antibodies increased bone density toa similar degree as PTH.

An additional study was conducted with the rat 11H10 bin antibody in arat closed fracture healing model. The fully rat 11H10 bin antibodyr11H10 was utilized in this study as a surrogate molecule to the fullyhuman antibodies described herein. The length of this study precludedthe use of the fully human DKK1 antibodies due to the rodent immuneresponse directed against human antibodies. Briefly a closed fracturewas generated in femur of 7-7.5 month old male rats (see methodology inExample 14). The femur was stabilized by insertion of a fine needle (18G) into the femur marrow space prior to fracture. The animals were thentreated with vehicle or r11H10 (25 mg/kg two times per week). Fractureswere allowed to heal for seven weeks. At the completion of the study thefractured femur was analyzed for bone mineral density, biomechanicalstrength and bridging. Anti-DKK1 treated animals showed significantimprovement in all these parameters, indicating that Anti-DKK1 therapywill be useful for the treatment of fracture healing, and otherindications where bone regeneration is needed. FIG. 6 shows theimprovement in maximum load and BMD achieved with Anti-DKK1 treatment atthe fracture callus indicating the acceleration of fracture healing.

Example 13 Detection of DKK1 in Human and Animal Model Serum and TissueSamples

The antibodies described herein have been used to detect DKK1 levels inhuman samples, including but not limited to serum. To develop this typeof assay it was important that two antibodies be selected that did notrecognize the same epitope, such as the two distinct epitopes describedherein. To assay for DKK1 in human serum or other tissues a standardcurve was first established using recombinant huDKK1. It was preferablethat this standard curve was established in human serum lacking, orcontaining low levels, of huDKK1. Typically the range of the standardcurve we use for serum is between 25 pg/ml and 10 ng/ml huDKK1, althoughthis range may need to be adjusted depending upon the minimal andmaximal values of huDKK1 obtained in the samples being analyzed. Anexample of the protocol used is as follows, but modifications obvious tothose skilled in the art may be taken depending upon the specificantibodies and samples utilized.

First the human serum to be analyzed was loaded into a non-bindinghalf-area plate. A predetermined amount of biotinylated-antibody fromepitope X (such as 11H10) and a predetermined amount of horseradishperoxidase (HRP) labeled antibody from epitope Y (such as 5.25.1) wasadded to the well with 50 mg/ml Rabbit IgG in I-block buffer to reach atotal volume in the well including serum of 60 μl. This mixture wasplaced on a shaker for 30 minutes and then incubated at 4° C. overnight.

Following the overnight incubation 50 μl of solution was transferredinto a 396 well plate. This plate was then incubated for 1 hour at roomtemperature with mixing. The well was washed with PBS and a detectionsolution was added. The plate was then analyzed on an appropriatereader. The assay was run in duplicate and concentration of DKK1 inserum was determined by comparison with the standard curve. The data areuseful in determining whether patients have altered DKK1 levels in thetissue or serum sample being analyzed.

In addition to using the antibodies described herein for the detectionof human DKK1 in human serum, the antibodies can also be used to detectDKK1 in serum isolated from animal models of disease. As a non-inclusiveexample, the protocol as described above was used to detect DKK1 levelsin a rat chronic kidney disease (CKD) model. An extract of diseased andcontrols kidneys were prepared and the level of rat DKK1 protein wasdetermined. The data are shown in FIG. 7 and demonstrate that DKK1protein levels are approximately five-fold elevated at 3-weeks afterinduction of kidney damage with the pharmacological agent. These resultsindicate that DKK1 is involved in the progression of kidney disease andsuggest that that pharmacological modulation of DKK1 is of therapeuticutility in kidney disease. Likewise, the methods describes in thisexample can be used to identify other disease states where DKK1modulation may have therapeutic utility.

Example 14

Sclerostin and DKK1 are negative regulators of bone formation.Inhibition of sclerostin by systemic treatment with a sclerostinmonoclonal antibody (Scl-Ab) significantly increased bone formation,bone mass and bone strength in animal models of osteoporosis (Li X D, etal. J Bone Miner Res 2009; 24:578). Furthermore, treatment with Scl-Abenhanced fracture healing in animal models of bone repair (Ke H Z, etal. Trans ORS 2009; 34:22; Ominsky M, et al. ASBMR abstract September2009; Denver, Colo.). Similarly, neutralization of DKK1 by systemicadministration of the monoclonal antibody r11H10 (DKK1-Ab) increasedbone mineral density (BMD) and strength at the fracture sites of mouse(Komatsu D E, et al. J Orthop Res 2010; DOI 10.1002/JOR.21078) and ratfracture models. We hypothesized that combination of Scl-Ab and DKK1-Abmay have a synergetic effect on stimulating bone formation andincreasing bone strength in fractured and non-fractured bone in an adultrat model.

Study Design: Seven to 7.5 months old male Sprague-Dawley (SD) rats(mean body weight 580 g) underwent unilateral closed femoralmid-diaphyseal fracture as reported previously (Bonnarens F, et al. JOrthop Res 1984; 2: 97-101). Briefly, an 18 gauge syringe needle wasinserted into the medullary canal through the femoral condyles, andserved as an internal fixation. The femur then underwent transversefracture via blunt impact loading at the anterior (lateral) aspect ofthe thigh. One day after fracture, animals (n=14-18/group) weresubcutaneously injected with either saline vehicle or Scl-Ab, or DKK1-Ab(r11H10) or combination of Scl-Ab and DKK1-Ab (Combination). Both Scl-Aband DKK1-Ab were given by subcutaneous injection at 25 mg/kg twice perweek. At 7 weeks post-fracture, animals were euthanized; the fracturedand intact, contralateral (CL) femurs were collected for densitometryand biomechanics. This study was approved by Amgen's Institution AnimalCare and Use Committee.

Densitometry: Femurs were scanned ex vivo by DXA (GE Lunar PIXImus II)at the fracture region (mid 30% of the fractured femur) or thecorresponding region in the CL femur to determine areal bone mineraldensity (BMD). Both femurs were also scanned using a desktop micro-CTsystem (eXplore Locus SP, GE Healthcare, London, Ontario, Canada) andreconstructed to a resolution of 30 μm. Bone mineral content (BMC,threshold of 800 mg/cc) of the central 1 mm of the fracture callus wereassessed after subtraction of the original cortex as previouslydescribed (Taylor D K, et al. J Bone Miner Res 2009; 24:1043-1054).Callus bone volume as a percent of total volume (BV/TV) was quantifiedusing a variable threshold (570 mg/cc for vehicle, Scl-Ab and DKK1-Ab;615 mg/cc for Combination). For the intact CL femur, regions spanning10% of the femur height at the midshaft (threshold 800 mg/cc) forcortical bone and distal femur trabecular bone (threshold 450 mg/cc forvehicle and DKK1-Ab, 550 mg/cc for Scl-Ab, and 600 mg/cc forcombination) were examined. Average cortical bone area and cancellousbone volume fraction (BV/TV) were assessed at these sites, respectively.

Biomechanics: Femurs were tested in 3-point bending to failure at thecenter of the fracture callus or at the midshaft of contralateral femur,and bone strength parameters were assessed (MTS 858 Mini Bionix II; spanlength=20 mm; displacement rate=0.1 mm/sec).

Statistical analyses: GraphPad Prism (v. 5.01) was used to perform thestatistic analysis. Group variances were compared by F test. If thegroup variances were significantly heterogeneous (p≤0.05), the data werelog-transformed and resubmitted assess the variance. When differencesbetween group variances were not significant, an unpaired t-test wasused to perform the group mean comparisons between the vehicle andScl-Ab or DKK1-Ab. When group variances remained heterogeneous (p≤0.05),then the comparison was conducted using the Mann Whitney test. Datareported as Mean+SE, and p<0.05 considered as significance.

RESULTS: Fractured femurs: both Scl-Ab and DKK1-Ab showed similarimprovement of bone mass and bone strength at fractured callus, asdemonstrated by an 11% increase at diaphyseal BMD by DXA, and a 24-26%increase at BMC by μCT and a 40-60% increase in BV/TV by μCT at central1 mm of fracture callus; and a 76-122% increase in peak load offractured bone, respectively, compared with vehicle. Combinationtreatment of Scl-Ab and DKK1-Ab greatly enhanced bone mass and bonestrength at fractured callus, to levels significantly greater thaneither one alone. Compared with vehicle, there was a 39%, 60% and 93%increase in diaphyseal BMD, BMC and BV/TV at central 1 mm of fracturecallus, respectively, in the Combination group. These changes led to a230% increase in peak load in the Combination group as compared withvehicle. In addition, BMD, BMC and BV/TV, and peak load weresignificantly higher in the Combination group compared with Scl-Ab aloneor DKK1-Ab alone groups.

Intact Contralateral Femurs:

DKK1-Ab did not significantly affect diaphyseal BMD, cortical bone areaand cancellous bone BV/TV and bone strength in intact contralateralfemurs. However, Scl-Ab significantly increased mid-diaphyseal corticalbone BMD by 6% and cortical bone area by 10%, and distal femurcancellous bone BV/TV by 43%, respectively, compared with vehicle. Theseincreases in bone mass at both cortical and cancellous bone sites underScl-Ab treatments associated with a 17% increase in peak load comparedwith vehicle.

Similar to the fractured bone, combination of Scl-Ab and DKK1-Absignificantly increased contralateral femoral mid-diaphyseal BMD by 12%,cortical bone area by 17% and distal femur cancellous bone BV/TV by107%, and peak load by 27% compared with vehicle. The mean values fordiaphyseal BMD and distal femur cancellous BV/TV in Combination groupwere significantly greater than those observed for the Scl-Ab andDKK1-Ab alone groups, while cortical area and peak load in theCombination group were significantly greater by 15% and 21% than theDKK1-Ab alone group, respectively.

What is claimed:
 1. An isolated antibody or fragment thereof thatspecifically binds to an epitope comprising amino acids 98-104 of SEQ IDNO:2.
 2. The antibody or fragment of claim 1, wherein the epitopefurther comprises amino acids 114-138 of SEQ ID NO:
 2. 3. The antibodyor fragment of claim 3 that binds to human DKK1 with a Kd of1.30E-03^(−s) or less.
 4. The antibody or fragment of claim 1 thatincreases bone mineral density in a patient.
 5. The antibody or fragmentof claim 1 that cross blocks the binding of a reference antibody tohuman DKK1 and/or is cross-blocked from binding to human DKK1 by thereference antibody, wherein the reference antibody comprises six CDRs,wherein the six CDRs comprise the amino acid sequences of SEQ ID NOs:133-138, 157-162, 163-168, 175-180, or 181-186.
 6. The antibody orfragment of claim 1, wherein the reference antibody comprises a heavychain variable region comprising the amino acid sequence of SEQ ID NO:36, 44, 52, 56, 64, or
 68. 7. The antibody or fragment of claim 1,wherein the reference antibody comprises a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 34, 42, 50, 54, 62, or66.
 8. The antibody or fragment of claim 7, wherein the referenceantibody comprises a light chain variable region and a heavy chainvariable region comprising the amino acid sequences of SEQ ID NOs: 34,and 36, respectively; 42 and 44, respectively; 50 and 52, respectively;54, and 56, respectively; 62 and 64, respectively; or 66 and 68,respectively.
 9. The antibody or fragment of claim 7, wherein thereference antibody is antibody 5.17.1, 5.25.1, 5.32.1, 5.40.1, 5.76.1,or 5.77.1, as depicted in Table
 1. 10. A method of treating a bonedisorder comprising administering an antibody or fragment of claim 1.11. The method of claim 10, wherein the bone disorder is a fracture. 12.A pharmaceutical composition comprising the antibody or fragment ofclaim
 1. 13. The antibody or fragment thereof of claim 1 in combinationwith one or more of a pharmaceutically acceptable excipient, diluent orcarrier.
 14. A method of accelerating bone fracture repair comprisingadministering to a patient in need thereof a combination of a DKK1antibody or fragment of claim 1 and a sclerostin antibody.
 15. Themethod of claim 14, wherein the antibodies are administeredcontemporaneously.
 16. The method of claim 15, wherein the antibodiesare administered within one day of the fracture.